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Sample records for protein complex connectivity

  1. Protein Connectivity in Chemotaxis Receptor Complexes

    PubMed Central

    Eismann, Stephan; Endres, Robert G.

    2015-01-01

    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. PMID:26646441

  2. Proteins Connecting the Nuclear Pore Complex with the Nuclear Interior

    PubMed Central

    Strambio-de-Castillia, Caterina; Blobel, Günter; Rout, Michael P.

    1999-01-01

    While much has been learned in recent years about the movement of soluble transport factors across the nuclear pore complex (NPC), comparatively little is known about intranuclear trafficking. We isolated the previously identified Saccharomyces protein Mlp1p (myosin-like protein) by an assay designed to find nuclear envelope (NE) associated proteins that are not nucleoporins. We localized both Mlp1p and a closely related protein that we termed Mlp2p to filamentous structures stretching from the nucleoplasmic face of the NE into the nucleoplasm, similar to the homologous vertebrate and Drosophila Tpr proteins. Mlp1p can be imported into the nucleus by virtue of a nuclear localization sequence (NLS) within its COOH-terminal domain. Overexpression experiments indicate that Mlp1p can form large structures within the nucleus which exclude chromatin but appear highly permeable to proteins. Remarkably, cells harboring a double deletion of MLP1 and MLP2 were viable, although they showed a slower net rate of active nuclear import and faster passive efflux of a reporter protein. Our data indicate that the Tpr homologues are not merely NPC-associated proteins but that they can be part of NPC-independent, peripheral intranuclear structures. In addition, we suggest that the Tpr filaments could provide chromatin-free conduits or tracks to guide the efficient translocation of macromolecules between the nucleoplasm and the NPC. PMID:10085285

  3. The Centrosomal Protein Pericentrin Identified at the Basal Body Complex of the Connecting Cilium in Mouse Photoreceptors

    PubMed Central

    Mühlhans, Johanna; Brandstätter, Johann Helmut; Gießl, Andreas

    2011-01-01

    Background Pericentrin (Pcnt), a conserved protein of the pericentriolar material, serves as a multifunctional scaffold for numerous proteins and plays an important role in microtubule organization. Recent studies indicate that Pcnt mutations are associated with a range of diseases including primordial dwarfism and ciliopathies. To date, three Pcnt splice variants from orthologous genes in mice and humans are known. Principal Findings We generated a specific Pcnt antiserum detecting all known Pcnt splice variants and examined the cellular and subcellular distribution of Pcnt in ciliated tissues of the mouse, the olfactory epithelium and the retina. For the first time, we identified Pcnt and its centrosomal interaction partners at the basal body complex of mouse retinal photoreceptors. Photoreceptors are morphologically and functionally subdivided into the light sensitive outer segment and the inner segment comprising the metabolic function of the cell. The two compartments are linked via a modified, specialized, non-motile cilium, the connecting cilium. Here, Pcnt colocalized with the whole protein machinery responsible for transport processes between the two compartments. Surprisingly, photoreceptors expressed a small Pcnt splice transcript – most likely a modified variant of Pcnt S – which was not present in receptor neurons of the olfactory epithelium. Conclusions Our findings suggest distinct functional roles of several Pcnt variants in different ciliated tissues and sensory neurons, like the olfactory epithelium and the retina of the mouse. The individual patchwork of different Pcnt splice transcripts seems to reflect the complexity of Pcnt function, an assumption corroborated by the heterogeneous clinical manifestations associated with mutations in the Pcnt gene. PMID:22031837

  4. Heterochromatin protein 1 (HP1) connects the FACT histone chaperone complex to the phosphorylated CTD of RNA polymerase II

    PubMed Central

    Kwon, So Hee; Florens, Laurence; Swanson, Selene K.; Washburn, Michael P.; Abmayr, Susan M.; Workman, Jerry L.

    2010-01-01

    Heterochromatin protein 1 (HP1) is well known as a silencing protein found at pericentric heterochromatin. Most eukaryotes have at least three isoforms of HP1 that play differential roles in heterochromatin and euchromatin. In addition to its role in heterochromatin, HP1 proteins have been shown to function in transcription elongation. To gain insights into the transcription functions of HP1, we sought to identify novel HP1-interacting proteins. Biochemical and proteomic approaches revealed that HP1 interacts with the histone chaperone complex FACT (facilitates chromatin transcription). HP1c interacts with the SSRP1 (structure-specific recognition protein 1) subunit and the intact FACT complex. Moreover, HP1c guides the recruitment of FACT to active genes and links FACT to active forms of RNA polymerase II. The absence of HP1c partially impairs the recruitment of FACT into heat-shock loci and causes a defect in heat-shock gene expression. Thus, HP1c functions to recruit the FACT complex to RNA polymerase II. PMID:20889714

  5. Protein Complexes in Bacteria

    PubMed Central

    Caufield, J. Harry; Abreu, Marco; Wimble, Christopher; Uetz, Peter

    2015-01-01

    Large-scale analyses of protein complexes have recently become available for Escherichia coli and Mycoplasma pneumoniae, yielding 443 and 116 heteromultimeric soluble protein complexes, respectively. We have coupled the results of these mass spectrometry-characterized protein complexes with the 285 “gold standard” protein complexes identified by EcoCyc. A comparison with databases of gene orthology, conservation, and essentiality identified proteins conserved or lost in complexes of other species. For instance, of 285 “gold standard” protein complexes in E. coli, less than 10% are fully conserved among a set of 7 distantly-related bacterial “model” species. Complex conservation follows one of three models: well-conserved complexes, complexes with a conserved core, and complexes with partial conservation but no conserved core. Expanding the comparison to 894 distinct bacterial genomes illustrates fractional conservation and the limits of co-conservation among components of protein complexes: just 14 out of 285 model protein complexes are perfectly conserved across 95% of the genomes used, yet we predict more than 180 may be partially conserved across at least half of the genomes. No clear relationship between gene essentiality and protein complex conservation is observed, as even poorly conserved complexes contain a significant number of essential proteins. Finally, we identify 183 complexes containing well-conserved components and uncharacterized proteins which will be interesting targets for future experimental studies. PMID:25723151

  6. Pigment-protein complexes

    SciTech Connect

    Siegelman, H W

    1980-01-01

    The photosynthetically-active pigment protein complexes of procaryotes and eucaryotes include chlorophyll proteins, carotenochlorophyll proteins, and biliproteins. They are either integral components or attached to photosynthetic membranes. Detergents are frequently required to solubilize the pigment-protein complexes. The membrane localization and detergent solubilization strongly suggest that the pigment-protein complexes are bound to the membranes by hydrophobic interactions. Hydrophobic interactions of proteins are characterized by an increase in entropy. Their bonding energy is directly related to temperature and ionic strength. Hydrophobic-interaction chromatography, a relatively new separation procedure, can furnish an important method for the purification of pigment-protein complexes. Phycobilisome purification and properties provide an example of the need to maintain hydrophobic interactions to preserve structure and function.

  7. Refining the Structural Model of a Heterohexameric Protein Complex: Surface Induced Dissociation and Ion Mobility Provide Key Connectivity and Topology Information

    PubMed Central

    2015-01-01

    Toyocamycin nitrile hydratase (TNH) is a protein hexamer that catalyzes the hydration of toyocamycin to produce sangivamycin. The structure of hexameric TNH and the arrangement of subunits within the complex, however, have not been solved by NMR or X-ray crystallography. Native mass spectrometry (MS) clearly shows that TNH is composed of two copies each of the α, β, and γ subunits. Previous surface induced dissociation (SID) tandem mass spectrometry on a quadrupole time-of-flight (QTOF) platform suggests that the TNH hexamer is a dimer composed of two αβγ trimers; furthermore, the results suggest that α–β interact most strongly (Blackwell et al. Anal. Chem. 2011, 83, 2862–286521417466). Here, multiple complementary MS based approaches and homology modeling have been applied to refine the structure of TNH. Solution-phase organic solvent disruption coupled with native MS agrees with the previous SID results. By coupling surface induced dissociation with ion mobility mass spectrometry (SID/IM), further information on the intersubunit contacts and relative interfacial strengths are obtained. The results show that TNH is a dimer of αβγ trimers, that within the trimer the α, β subunits bind most strongly, and that the primary contact between the two trimers is through a γ–γ interface. Collisional cross sections (CCSs) measured from IM experiments are used as constraints for postulating the arrangement of the subunits represented by coarse-grained spheres. Covalent labeling (surface mapping) together with protein complex homology modeling and docking of trimers to form hexamer are utilized with all the above information to propose the likely quaternary structure of TNH, with chemical cross-linking providing cross-links consistent with the proposed structure. The novel feature of this approach is the use of SID-MS with ion mobility to define complete connectivity and relative interfacial areas of a heterohexameric protein complex, providing much more

  8. Making the Chromosome-Gene-Protein Connection.

    ERIC Educational Resources Information Center

    Mulvihill, Charlotte

    1996-01-01

    Presents an exercise that demonstrates the chromosome-gene-protein connection using sickle-cell anemia, a genetic disease with a well-characterized molecular basis. Involves connecting changes in DNA to protein outcomes and tying them into the next generation by meiosis and gamete formation with genetic crosses. Motivates students to integrate…

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

    PubMed Central

    2011-01-01

    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. PMID:22165991

  10. Proteins, fluctuations and complexity

    SciTech Connect

    Frauenfelder, Hans; Chen, Guo; Fenimore, Paul W

    2008-01-01

    Glasses, supercooled liquids, and proteins share common properties, in particular the existence of two different types of fluctuations, {alpha} and {beta}. While the effect of the {alpha} fluctuations on proteins has been known for a few years, the effect of {beta} fluctuations has not been understood. By comparing neutron scattering data on the protein myoglobin with the {beta} fluctuations in the hydration shell measured by dielectric spectroscopy we show that the internal protein motions are slaved to these fluctuations. We also show that there is no 'dynamic transition' in proteins near 200 K. The rapid increase in the mean square displacement with temperature in many neutron scattering experiments is quantitatively predicted by the {beta} fluctuations in the hydration shell.

  11. Protein-protein interaction networks (PPI) and complex diseases

    PubMed Central

    Safari-Alighiarloo, Nahid; Taghizadeh, Mohammad; Rezaei-Tavirani, Mostafa; Goliaei, Bahram

    2014-01-01

    The physical interaction of proteins which lead to compiling them into large densely connected networks is a noticeable subject to investigation. Protein interaction networks are useful because of making basic scientific abstraction and improving biological and biomedical applications. Based on principle roles of proteins in biological function, their interactions determine molecular and cellular mechanisms, which control healthy and diseased states in organisms. Therefore, such networks facilitate the understanding of pathogenic (and physiologic) mechanisms that trigger the onset and progression of diseases. Consequently, this knowledge can be translated into effective diagnostic and therapeutic strategies. Furthermore, the results of several studies have proved that the structure and dynamics of protein networks are disturbed in complex diseases such as cancer and autoimmune disorders. Based on such relationship, a novel paradigm is suggested in order to confirm that the protein interaction networks can be the target of therapy for treatment of complex multi-genic diseases rather than individual molecules with disrespect the network. PMID:25436094

  12. "Only Connect"? Complexities in International Student Communication

    ERIC Educational Resources Information Center

    Bell, Frances; Zaitseva, Elena

    2005-01-01

    This article explores the potential and limitations of international educational collaboration using the concept of "connection", a term with different meanings that are sometimes conflated to produce unrealistic expectations of computer-mediated communication (CMC). The authors explore the use on the Internet of the "only connect" quote from…

  13. Connectivity and complex systems in geomorphology: addressing some key challenges

    NASA Astrophysics Data System (ADS)

    Pöppl, Ronald; Turnbull-Lloyd, Laura; Parsons, Anthony; Bracken, Louise; Keesstra, Saskia; Masselink, Rens

    2016-04-01

    "Connectivity thinking" and related concepts have a long history in geomorphology. Since the beginning of the 21st century connectivity research experienced a huge boom in geomorphology as geomorphologists started to develop new concepts on connectivity to better understand the complexity of geomorphic systems and system response to change. However, progress in the field of connectivity in geomorphology has mostly been developing in a parallel manner, resulting in a multiplicity of definitions, concepts and methodological approaches. Nevertheless, a set of common key challenges amongst the different connectivity concepts and approaches used to understand complex geomorphic systems are also evident. In the course of a theory think tank of the COST Action ES1306 (CONNECTEUR - Connecting European Connectivity Research) the following five different key challenges were detected (Turnbull et al., in prep.): (i) defining the fundamental unit, (ii) distinguishing between structural and functional boundaries, (iii) emergent behavior, (iv) memory effects, (v) measuring connectivity. In this presentation we will a) discuss how these key challenges are addressed and approached in connectivity research in geomorphology, b) evaluate ways in which cross-disciplinary advances may be made by exploring potential for a common toolbox approach to the study of connectivity.

  14. Protein sorting in complex plastids.

    PubMed

    Sheiner, Lilach; Striepen, Boris

    2013-02-01

    Taming a cyanobacterium in a pivitol event of endosymbiosis brought photosynthesis to eukaryotes, and gave rise to the plastids found in glaucophytes, red and green algae, and the descendants of the latter, the plants. Ultrastructural as well as molecular research over the last two decades has demonstrated that plastids have enjoyed surprising lateral mobility across the tree of life. Numerous independent secondary and tertiary endosymbiosis have led to a spread of plastids into a variety of, up to that point, non-photosynthetic lineages. Happily eating and subsequently domesticating one another protists conquered a wide variety of ecological niches. The elaborate evolution of secondary, or complex, plastids is reflected in the numerous membranes that bound them (three or four compared to the two membranes of the primary plastids). Gene transfer to the host nucleus is a hallmark of endosymbiosis and provides centralized cellular control. Here we review how these proteins find their way back into the stroma of the organelle and describe the advances in the understanding of the molecular mechanisms that allow protein translocation across four membranes. This article is part of a Special Issue entitled: Protein Import and Quality Control in Mitochondria and Plastids.

  15. Dynamic interactions of proteins in complex networks

    SciTech Connect

    Appella, E.; Anderson, C.

    2009-10-01

    evidence indicates that disordered domains can acquire structural features that modulate the binding and strength of the signaling cascade. Whereas the first two minireviews describe ways in which protein interactions are modulated, the third, by Tompa, focuses on the importance of protein disorder in a subset of amyloid proteins. It is apparent that within this group, part of the polypeptide chain remains disordered during amyloid formation. Moreover, the disordered segments have different amino acid composition and physicochemical characteristics, which suggests that they may play a role in amyloid stability. The disordered region may serve as a linker to connect the ordered core and a globular domain, maintaining the stability and structure of the globular domain and minimizing protein refolding upon amyloid formation. As techniques in protein chemistry advance, we are learning more and more about the mechanisms that regulate and are regulated by protein interactions. The three minireviews in this series offer a glimpse of the complex dynamics fundamental to protein-protein interactions. In the future, we expect that the knowledge gained will help to augment our ability to control complex pathologies and treat diverse diseases states.

  16. Structure Prediction of Protein Complexes

    NASA Astrophysics Data System (ADS)

    Pierce, Brian; Weng, Zhiping

    Protein-protein interactions are critical for biological function. They directly and indirectly influence the biological systems of which they are a part. Antibodies bind with antigens to detect and stop viruses and other infectious agents. Cell signaling is performed in many cases through the interactions between proteins. Many diseases involve protein-protein interactions on some level, including cancer and prion diseases.

  17. Connectivity independent protein-structure alignment: a hierarchical approach

    PubMed Central

    Kolbeck, Bjoern; May, Patrick; Schmidt-Goenner, Tobias; Steinke, Thomas; Knapp, Ernst-Walter

    2006-01-01

    Background Protein-structure alignment is a fundamental tool to study protein function, evolution and model building. In the last decade several methods for structure alignment were introduced, but most of them ignore that structurally similar proteins can share the same spatial arrangement of secondary structure elements (SSE) but differ in the underlying polypeptide chain connectivity (non-sequential SSE connectivity). Results We perform protein-structure alignment using a two-level hierarchical approach implemented in the program GANGSTA. On the first level, pair contacts and relative orientations between SSEs (i.e. α-helices and β-strands) are maximized with a genetic algorithm (GA). On the second level residue pair contacts from the best SSE alignments are optimized. We have tested the method on visually optimized structure alignments of protein pairs (pairwise mode) and for database scans. For a given protein structure, our method is able to detect significant structural similarity of functionally important folds with non-sequential SSE connectivity. The performance for structure alignments with strictly sequential SSE connectivity is comparable to that of other structure alignment methods. Conclusion As demonstrated for several applications, GANGSTA finds meaningful protein-structure alignments independent of the SSE connectivity. GANGSTA is able to detect structural similarity of protein folds that are assigned to different superfamilies but nevertheless possess similar structures and perform related functions, even if these proteins differ in SSE connectivity. PMID:17118190

  18. Mass Spectrometry of Intact Membrane Protein Complexes

    PubMed Central

    Laganowsky, Arthur; Reading, Eamonn; Hopper, Jonathan T.S.; Robinson, Carol V.

    2014-01-01

    Mass spectrometry of intact soluble protein complexes has emerged as a powerful technique to study the stoichiometry, structure-function and dynamics of protein assemblies. Recent developments have extended this technique to the study of membrane protein complexes where it has already revealed subunit stoichiometries and specific phospholipid interactions. Here, we describe a protocol for mass spectrometry of membrane protein complexes. The protocol begins with preparation of the membrane protein complex enabling not only the direct assessment of stoichiometry, delipidation, and quality of the target complex, but also evaluation of the purification strategy. A detailed list of compatible non-ionic detergents is included, along with a protocol for screening detergents to find an optimal one for mass spectrometry, biochemical and structural studies. This protocol also covers the preparation of lipids for protein-lipid binding studies and includes detailed settings for a Q-ToF mass spectrometer after introduction of complexes from gold-coated nanoflow capillaries. PMID:23471109

  19. Investigation of a protein complex network

    NASA Astrophysics Data System (ADS)

    Mashaghi, A. R.; Ramezanpour, A.; Karimipour, V.

    2004-09-01

    The budding yeast Saccharomyces cerevisiae is the first eukaryote whose genome has been completely sequenced. It is also the first eukaryotic cell whose proteome (the set of all proteins) and interactome (the network of all mutual interactions between proteins) has been analyzed. In this paper we study the structure of the yeast protein complex network in which weighted edges between complexes represent the number of shared proteins. It is found that the network of protein complexes is a small world network with scale free behavior for many of its distributions. However we find that there are no strong correlations between the weights and degrees of neighboring complexes. To reveal non-random features of the network we also compare it with a null model in which the complexes randomly select their proteins. Finally we propose a simple evolutionary model based on duplication and divergence of proteins.

  20. Structural Studies of Protein-Surfactant Complexes

    SciTech Connect

    Chodankar, S. N.; Aswal, V. K.; Wagh, A. G.

    2008-03-17

    The structure of protein-surfactant complexes of two proteins bovine serum albumin (BSA) and lysozyme in presence of anionic surfactant sodium dodecyl sulfate (SDS) has been studied using small-angle neutron scattering (SANS). It is observed that these two proteins form different complex structures with the surfactant. While BSA protein undergoes unfolding on addition of surfactant, lysozyme does not show any unfolding even up to very high surfactant concentrations. The unfolding of BSA protein is caused by micelle-like aggregation of surfactant molecules in the complex. On the other hand, for lysozyme protein there is only binding of individual surfactant molecules to protein. Lysozyme in presence of higher surfactant concentrations has protein-surfactant complex structure coexisting with pure surfactant micelles.

  1. Protein import into complex plastids: Cellular organization of higher complexity.

    PubMed

    Maier, Uwe G; Zauner, Stefan; Hempel, Franziska

    2015-01-01

    Many protists with high ecological and medical relevance harbor plastids surrounded by four membranes. Thus, nucleus-encoded proteins of these complex plastids have to traverse these barriers. Here we report on the identification of the protein translocators located in two of the plastid surrounding membranes and present recent findings on the mechanisms of protein import into the plastids of diatoms.

  2. A Protein Complex Map of Trypanosoma brucei

    PubMed Central

    Mehta, Vaibhav; Najafabadi, Hamed S.; Moshiri, Houtan; Jardim, Armando; Salavati, Reza

    2016-01-01

    The functions of the majority of trypanosomatid-specific proteins are unknown, hindering our understanding of the biology and pathogenesis of Trypanosomatida. While protein-protein interactions are highly informative about protein function, a global map of protein interactions and complexes is still lacking for these important human parasites. Here, benefiting from in-depth biochemical fractionation, we systematically interrogated the co-complex interactions of more than 3354 protein groups in procyclic life stage of Trypanosoma brucei, the protozoan parasite responsible for human African trypanosomiasis. Using a rigorous methodology, our analysis led to identification of 128 high-confidence complexes encompassing 716 protein groups, including 635 protein groups that lacked experimental annotation. These complexes correlate well with known pathways as well as for proteins co-expressed across the T. brucei life cycle, and provide potential functions for a large number of previously uncharacterized proteins. We validated the functions of several novel proteins associated with the RNA-editing machinery, identifying a candidate potentially involved in the mitochondrial post-transcriptional regulation of T. brucei. Our data provide an unprecedented view of the protein complex map of T. brucei, and serve as a reliable resource for further characterization of trypanosomatid proteins. The presented results in this study are available at: www.TrypsNetDB.org. PMID:26991453

  3. Co-translational assembly of protein complexes.

    PubMed

    Wells, Jonathan N; Bergendahl, L Therese; Marsh, Joseph A

    2015-12-01

    The interaction of biological macromolecules is a fundamental attribute of cellular life. Proteins, in particular, often form stable complexes with one another. Although the importance of protein complexes is widely recognized, we still have only a very limited understanding of the mechanisms underlying their assembly within cells. In this article, we review the available evidence for one such mechanism, namely the coupling of protein complex assembly to translation at the polysome. We discuss research showing that co-translational assembly can occur in both prokaryotic and eukaryotic organisms and can have important implications for the correct functioning of the complexes that result. Co-translational assembly can occur for both homomeric and heteromeric protein complexes and for both proteins that are translated directly into the cytoplasm and those that are translated into or across membranes. Finally, we discuss the properties of proteins that are most likely to be associated with co-translational assembly.

  4. Complex Reconstitution from Individual Protein Modules.

    PubMed

    Basquin, Jérôme; Taschner, Michael; Lorentzen, Esben

    2016-01-01

    Cellular function relies on protein complexes that work as nano-machines. The structure and function of protein complexes is an outcome of the specific combination of protein subunits, or modules, within the complex. A major focus of molecular biology is thus to understand how protein subunits assemble to form complexes with distinct biological function. To this end, in vitro reconstitution of complexes from individual subunits to study their assembly, structure and activity is of central importance. With purified individual subunits and sub-modules at hand one can systematically dissect the hierarchical assembly of larger complexes using direct protein-protein interaction assays. Furthermore, activity assays can be carried out with individual subunits or smaller sub-complexes and compared to those of the fully assembled complex to precisely map functional sites and provide a molecular basis for in vivo observations. In this chapter we review methods for protein complex assembly from individual subunits and provide examples of advantages and potential pitfalls to this approach. PMID:27165333

  5. Fish utilisation of wetland nurseries with complex hydrological connectivity.

    PubMed

    Davis, Ben; Johnston, Ross; Baker, Ronald; Sheaves, Marcus

    2012-01-01

    The physical and faunal characteristics of coastal wetlands are driven by dynamics of hydrological connectivity to adjacent habitats. Wetlands on estuary floodplains are particularly dynamic, driven by a complex interplay of tidal marine connections and seasonal freshwater flooding, often with unknown consequences for fish using these habitats. To understand the patterns and subsequent processes driving fish assemblage structure in such wetlands, we examined the nature and diversity of temporal utilisation patterns at a species or genus level over three annual cycles in a tropical Australian estuarine wetland system. Four general patterns of utilisation were apparent based on CPUE and size-structure dynamics: (i) classic nursery utlisation (use by recently settled recruits for their first year) (ii) interrupted peristence (iii) delayed recruitment (iv) facultative wetland residence. Despite the small self-recruiting 'facultative wetland resident' group, wetland occupancy seems largely driven by connectivity to the subtidal estuary channel. Variable connection regimes (i.e. frequency and timing of connections) within and between different wetland units (e.g. individual pools, lagoons, swamps) will therefore interact with the diversity of species recruitment schedules to generate variable wetland assemblages in time and space. In addition, the assemblage structure is heavily modified by freshwater flow, through simultaneously curtailing persistence of the 'interrupted persistence' group, establishing connectivity for freshwater spawned members of both the 'facultative wetland resident' and 'delayed recruitment group', and apparently mediating use of intermediate nursery habitats for marine-spawned members of the 'delayed recruitment' group. The diversity of utilisation pattern and the complexity of associated drivers means assemblage compositions, and therefore ecosystem functioning, is likely to vary among years depending on variations in hydrological connectivity

  6. Fish Utilisation of Wetland Nurseries with Complex Hydrological Connectivity

    PubMed Central

    Davis, Ben; Johnston, Ross; Baker, Ronald; Sheaves, Marcus

    2012-01-01

    The physical and faunal characteristics of coastal wetlands are driven by dynamics of hydrological connectivity to adjacent habitats. Wetlands on estuary floodplains are particularly dynamic, driven by a complex interplay of tidal marine connections and seasonal freshwater flooding, often with unknown consequences for fish using these habitats. To understand the patterns and subsequent processes driving fish assemblage structure in such wetlands, we examined the nature and diversity of temporal utilisation patterns at a species or genus level over three annual cycles in a tropical Australian estuarine wetland system. Four general patterns of utilisation were apparent based on CPUE and size-structure dynamics: (i) classic nursery utlisation (use by recently settled recruits for their first year) (ii) interrupted peristence (iii) delayed recruitment (iv) facultative wetland residence. Despite the small self-recruiting ‘facultative wetland resident’ group, wetland occupancy seems largely driven by connectivity to the subtidal estuary channel. Variable connection regimes (i.e. frequency and timing of connections) within and between different wetland units (e.g. individual pools, lagoons, swamps) will therefore interact with the diversity of species recruitment schedules to generate variable wetland assemblages in time and space. In addition, the assemblage structure is heavily modified by freshwater flow, through simultaneously curtailing persistence of the ’interrupted persistence’ group, establishing connectivity for freshwater spawned members of both the ‘facultative wetland resident’ and ‘delayed recruitment group’, and apparently mediating use of intermediate nursery habitats for marine-spawned members of the ‘delayed recruitment’ group. The diversity of utilisation pattern and the complexity of associated drivers means assemblage compositions, and therefore ecosystem functioning, is likely to vary among years depending on variations in

  7. 3D complex: a structural classification of protein complexes.

    PubMed

    Levy, Emmanuel D; Pereira-Leal, Jose B; Chothia, Cyrus; Teichmann, Sarah A

    2006-11-17

    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.

  8. Protein complexes that control renal epithelial polarity

    PubMed Central

    Pieczynski, Jay

    2011-01-01

    Establishment of epithelial apicobasal polarity is crucial for proper kidney development and function. In recent years, there have been important advances in our understanding of the factors that mediate the initiation of apicobasal polarization. Key among these are the polarity complexes that are evolutionarily conserved from simple organisms to humans. Three of these complexes are discussed in this review: the Crumbs complex, the Par complex, and the Scribble complex. The apical Crumbs complex consists of three proteins, Crumbs, PALS1, and PATJ, whereas the apical Par complex consists of Par-3, Par-6, and atypical protein kinase C. The lateral Scribble complex consists of Scribble, discs large, and lethal giant larvae. These complexes modulate kinase and small G protein activity such that the apical and basolateral complexes signal antagonistically, leading to the segregation of the apical and basolateral membranes. The polarity complexes also serve as scaffolds to direct and retain proteins at the apical membrane, the basolateral membrane, or the intervening tight junction. There is plasticity in apicobasal polarity, and this is best seen in the processes of epithelial-to-mesenchymal transition and the converse mesenchymal-to-epithelial transition. These transitions are important in kidney disease as well as kidney development, and modulation of the polarity complexes are critical for these transitions. PMID:21228104

  9. Integrating Mass Spectrometry of Intact Protein Complexes into Structural Proteomics

    PubMed Central

    Hyung, Suk-Joon; Ruotolo, Brandon T.

    2013-01-01

    Summary Mass spectrometry analysis of intact protein complexes has emerged as an established technology for assessing the composition and connectivity within dynamic, heterogeneous multiprotein complexes at low concentrations and in the context of mixtures. As this technology continues to move forward, one of the main challenges is to integrate the information content of such intact protein complex measurements with other mass spectrometry approaches in structural biology. Methods such as H/D exchange, oxidative foot-printing, chemical cross-linking, affinity purification, and ion mobility separation add complementary information that allows access to every level of protein structure and organization. Here, we survey the structural information that can be retrieved by such experiments, demonstrate the applicability of integrative mass spectrometry approaches in structural proteomics, and look to the future to explore upcoming innovations in this rapidly-advancing area. PMID:22611037

  10. Complementary Proteomic Analysis of Protein Complexes

    PubMed Central

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

    2013-01-01

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

  11. The Claudin Megatrachea Protein Complex*

    PubMed Central

    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

  12. Interaction proteomics of synapse protein complexes

    PubMed Central

    Klemmer, Patricia; Smit, August B.

    2010-01-01

    The brain integrates complex types of information, and executes a wide range of physiological and behavioral processes. Trillions of tiny organelles, the synapses, are central to neuronal communication and information processing in the brain. Synaptic transmission involves an intricate network of synaptic proteins that forms the molecular machinery underlying transmitter release, activation, and modulation of transmitter receptors and signal transduction cascades. These processes are dynamically regulated and underlie neuroplasticity, crucial to learning and memory formation. In recent years, interaction proteomics has increasingly been used to elucidate the constituents of synaptic protein complexes. Unlike classic hypothesis-based assays, interaction proteomics detects both known and novel interactors without bias. In this trend article, we focus on the technical aspects of recent proteomics to identify synapse protein complexes, and the complementary methods used to verify the protein–protein interaction. Moreover, we discuss the experimental feasibility of performing global analysis of the synapse protein interactome. PMID:20361179

  13. [Protein-energy malnutrition in patients with connective tissue dysplasia].

    PubMed

    Lialiukova, E A

    2013-01-01

    In the conditions of the specialized Center of a dysplasia of a connecting tissue the assessment of an protein--energy malnutrition at 121 patients with signs of a dysplasia of a connecting tissue is carried out. High frequency of an oligotrophy at patients with a dysplasia of a connecting tissue is registered. The I degree of a gipotorofiya is taped at 26.21% of the patients, II degree--at 18.44%, the III degree--at 3.88% of patients.

  14. Connectivity and Excluded Volume Effects in Polymeric Complex Coacervates

    NASA Astrophysics Data System (ADS)

    Sing, Charles; Radhakrishna, Mithun

    Oppositely-charged polyelectrolytes in salt solutions can undergo phase separation to form complex coacervates. This charge-driven phase behavior is the basis for emerging motifs in self-assembly. Traditional uses for coacervates are in food and personal care products, while applications in technologies for drug delivery and sensory materials are being developed. One of the primary theories driving understanding of complex coacervates is the Voorn-Overbeek (V-O) theory, which is a precursor to more sophisticated field theories. We present both theory and simulation that provides an alternate picture of coacervates, specifically addressing the limitations of V-O. Our theoretical approach is based on PRISM, which is a liquid-state theory that specifically accounts for connectivity. This is compared with Monte Carlo-based simulations, which likewise provide a molecular picture of coacervation. We demonstrate that a combination of connectivity-based correlations and excluded volume has a profound effect on coacervation phase behavior, suggesting that favorable comparison of V-O to experiment benefits from a cancellation of errors. The influence of connectivity on coacervate phase behavior hints at new opportunities for molecular-based design in electrostatically-driven self-assembly.

  15. Complex coacervation of supercharged proteins with polyelectrolytes.

    PubMed

    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.

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

    PubMed

    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

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

    PubMed

    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.

  18. Optimization of the electrostatic interactions in protein-protein complexes

    NASA Astrophysics Data System (ADS)

    Alexov, Emil; Brock, Kelly; Kundrotas, Petras

    2007-03-01

    Electrostatic energy is one of the driving forces of protein-protein association. Understanding the role of the energy components on the energetics of protein-protein association will help us in engineering protein-protein interactions and could lead to development of scoring functions that can rank alternative models and decoys. Here we investigate whether the components of the electrostatic energy of protein-protein complexes is optimized in respect to random distribution of the charged residues. We report a clear tendency that coulombic electrostatic interactions are optimized, while the reaction field energy is inversely optimized. It was found that the maximum of the coulombic energy Z-score is shifted 3 units away from the origin and the maximum of the reaction field energy by 2 units. Such a large shift of the Z-score of both coulombic and reaction field energies indicates that wild-type protein-protein interactions are in most cases optimized in terms of coulombic interactions while compromising reaction field energy. Based on these finding a scoring function was developed as a linear combination of the Z-score of the coulombic interactions minus Z-score of the reaction field energy. The scoring function was tested against the decoy sets and it was shown that in majority of the cases we can identify the wild-type complex among hundreds of decoys.

  19. Measuring the functional sequence complexity of proteins

    PubMed Central

    Durston, Kirk K; Chiu, David KY; Abel, David L; Trevors, Jack T

    2007-01-01

    Background Abel and Trevors have delineated three aspects of sequence complexity, Random Sequence Complexity (RSC), Ordered Sequence Complexity (OSC) and Functional Sequence Complexity (FSC) observed in biosequences such as proteins. In this paper, we provide a method to measure functional sequence complexity. Methods and Results We have extended Shannon uncertainty by incorporating the data variable with a functionality variable. The resulting measured unit, which we call Functional bit (Fit), is calculated from the sequence data jointly with the defined functionality variable. To demonstrate the relevance to functional bioinformatics, a method to measure functional sequence complexity was developed and applied to 35 protein families. Considerations were made in determining how the measure can be used to correlate functionality when relating to the whole molecule and sub-molecule. In the experiment, we show that when the proposed measure is applied to the aligned protein sequences of ubiquitin, 6 of the 7 highest value sites correlate with the binding domain. Conclusion For future extensions, measures of functional bioinformatics may provide a means to evaluate potential evolving pathways from effects such as mutations, as well as analyzing the internal structural and functional relationships within the 3-D structure of proteins. PMID:18062814

  20. Intrinsically disordered proteins aggregate at fungal cell-to-cell channels and regulate intercellular connectivity

    PubMed Central

    Lai, Julian; Koh, Chuan Hock; Tjota, Monika; Pieuchot, Laurent; Raman, Vignesh; Chandrababu, Karthik Balakrishna; Yang, Daiwen; Wong, Limsoon; Jedd, Gregory

    2012-01-01

    Like animals and plants, multicellular fungi possess cell-to-cell channels (septal pores) that allow intercellular communication and transport. Here, using a combination of MS of Woronin body-associated proteins and a bioinformatics approach that identifies related proteins based on composition and character, we identify 17 septal pore-associated (SPA) proteins that localize to the septal pore in rings and pore-centered foci. SPA proteins are not homologous at the primary sequence level but share overall physical properties with intrinsically disordered proteins. Some SPA proteins form aggregates at the septal pore, and in vitro assembly assays suggest aggregation through a nonamyloidal mechanism involving mainly α-helical and disordered structures. SPA loss-of-function phenotypes include excessive septation, septal pore degeneration, and uncontrolled Woronin body activation. Together, our data identify the septal pore as a complex subcellular compartment and focal point for the assembly of unstructured proteins controlling diverse aspects of intercellular connectivity. PMID:22955885

  1. Protein-protein interactions in the synaptonemal complex.

    PubMed Central

    Tarsounas, M; Pearlman, R E; Gasser, P J; Park, M S; Moens, P B

    1997-01-01

    In mammalian systems, an approximately M(r) 30,000 Cor1 protein has been identified as a major component of the meiotic prophase chromosome cores, and a M(r) 125,000 Syn1 protein is present between homologue cores where they are synapsed and form the synaptonemal complex (SC). Immunolocalization of these proteins during meiosis suggests possible homo- and heterotypic interactions between the two as well as possible interactions with yet unrecognized proteins. We used the two-hybrid system in the yeast Saccharomyces cerevisiae to detect possible protein-protein associations. Segments of hamsters Cor1 and Syn1 proteins were tested in various combinations for homo- and heterotypic interactions. In the cause of Cor1, homotypic interactions involve regions capable of coiled-coil formation, observation confirmed by in vitro affinity coprecipitation experiments. The two-hybrid assay detects no interaction of Cor1 protein with central and C-terminal fragments of Syn1 protein and no homotypic interactions involving these fragments of Syn1. Hamster Cor1 and Syn1 proteins both associate with the human ubiquitin-conjugation enzyme Hsubc9 as well as with the hamster Ubc9 homologue. The interactions between SC proteins and the Ubc9 protein may be significant for SC disassembly, which coincides with the repulsion of homologs by late prophase I, and also for the termination of sister centromere cohesiveness at anaphase II. Images PMID:9285814

  2. Complex micropatterning of proteins within microfluidic channels.

    PubMed

    Kim, Miju; Doh, Junsang

    2014-01-01

    Microfluidic channels containing protein micropatterned surfaces are useful in many bioanalytical and biological applications. In this study, we developed a new method to integrate microfluidics and protein micropatterning by attaching poly(dimethylsiloxane) (PDMS) microfluidic channels to bio-friendly photoresist films via poly(dopamine) (PDA) adhesive. A bio-friendly photoresist poly(2,2-dimethoxy nitrobenzyl methacrylate-r-methyl methacrylate-r-poly(ethylene glycol) methacrylate) (PDMP) was synthesized and used. By performing microscope projection photolithography (MPP) to the PDMP thin films within PDMS microchannels, complex micropatterns of proteins were successfully generated within microfluidic channels. PMID:25570075

  3. New Anthocyanin-Human Salivary Protein Complexes.

    PubMed

    Ferrer-Gallego, Raúl; Soares, Susana; Mateus, Nuno; Rivas-Gonzalo, Julián; Escribano-Bailón, M Teresa; de Freitas, Victor

    2015-08-01

    The interaction between phenolic compounds and salivary proteins is considered the basis of the poorly understood phenomenon of astringency. Furthermore, this interaction is an important factor in relation to their bioavailability. In this work, interactions between anthocyanin and human salivary protein fraction were studied by mass spectrometry (MALDI-TOF-MS and FIA-ESI-MS) and saturation-transfer difference (STD) NMR spectroscopy. Anthocyanins were able to interact with saliva proteins. The dissociation constant (KD) between malvidin 3-glucoside and salivary proline-rich proteins was 1.92 mM for the hemiketal form (pH 3.4) and 1.83 mM for the flavylium cation (pH 1.0). New soluble complexes between these salivary proteins and malvidin 3-glucoside were identified for the first time.

  4. Peroxisome protein import: a complex journey

    PubMed Central

    Baker, Alison; Hogg, Thomas Lanyon; Warriner, Stuart L.

    2016-01-01

    The import of proteins into peroxisomes possesses many unusual features such as the ability to import folded proteins, and a surprising diversity of targeting signals with differing affinities that can be recognized by the same receptor. As understanding of the structure and function of many components of the protein import machinery has grown, an increasingly complex network of factors affecting each step of the import pathway has emerged. Structural studies have revealed the presence of additional interactions between cargo proteins and the PEX5 receptor that affect import potential, with a subtle network of cargo-induced conformational changes in PEX5 being involved in the import process. Biochemical studies have also indicated an interdependence of receptor–cargo import with release of unloaded receptor from the peroxisome. Here, we provide an update on recent literature concerning mechanisms of protein import into peroxisomes. PMID:27284042

  5. On protein abundance distributions in complex mixtures

    PubMed Central

    2013-01-01

    Mass spectrometry, an analytical technique that measures the mass-to-charge ratio of ionized atoms or molecules, dates back more than 100 years, and has both qualitative and quantitative uses for determining chemical and structural information. Quantitative proteomic mass spectrometry on biological samples focuses on identifying the proteins present in the samples, and establishing the relative abundances of those proteins. Such protein inventories create the opportunity to discover novel biomarkers and disease targets. We have previously introduced a normalized, label-free method for quantification of protein abundances under a shotgun proteomics platform (Griffin et al., 2010). The introduction of this method for quantifying and comparing protein levels leads naturally to the issue of modeling protein abundances in individual samples. We here report that protein abundance levels from two recent proteomics experiments conducted by the authors can be adequately represented by Sichel distributions. Mathematically, Sichel distributions are mixtures of Poisson distributions with a rather complex mixing distribution, and have been previously and successfully applied to linguistics and species abundance data. The Sichel model can provide a direct measure of the heterogeneity of protein abundances, and can reveal protein abundance differences that simpler models fail to show. PMID:23360617

  6. Troposphere-lower-stratosphere connection in an intermediate complexity model.

    NASA Astrophysics Data System (ADS)

    Ruggieri, Paolo; King, Martin; Kucharski, Fred; Buizza, Roberto; Visconti, Guido

    2016-04-01

    The dynamical coupling between the troposphere and the lower stratosphere has been investigated using a low-top, intermediate complexity model provided by the Abdus Salam International Centre for Theoretical Physics (SPEEDY). The key question that we wanted to address is whether a simple model like SPEEDY can be used to understand troposphere-stratosphere interactions, e.g. forced by changes of sea-ice concentration in polar arctic regions. Three sets of experiments have been performed. Firstly, a potential vorticity perspective has been applied to understand the wave-like forcing of the troposphere on the stratosphere and to provide quantitative information on the sub seasonal variability of the coupling. Then, the zonally asymmetric, near-surface response to a lower-stratospheric forcing has been analysed in a set of forced experiments with an artificial heating imposed in the extra-tropical lower stratosphere. Finally, the lower-stratosphere response sensitivity to tropospheric initial conditions has been examined. Results indicate how SPEEDY captures the physics of the troposphere-stratosphere connection but also show the lack of stratospheric variability. Results also suggest that intermediate-complexity models such as SPEEDY could be used to investigate the effects that surface forcing (e.g. due to sea-ice concentration changes) have on the troposphere and the lower stratosphere.

  7. Isolation of proteins and protein complexes by immunoprecipitation.

    PubMed

    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.

  8. Connections between cadherin-catenin proteins, spindle misorientation, and cancer

    PubMed Central

    Shahbazi, Marta N; Perez-Moreno, Mirna

    2015-01-01

    Cadherin-catenin mediated adhesion is an important determinant of tissue architecture in multicellular organisms. Cancer progression and maintenance is frequently associated with loss of their expression or functional activity, which not only leads to decreased cell-cell adhesion, but also to enhanced tumor cell proliferation and loss of differentiated characteristics. This review is focused on the emerging implications of cadherin-catenin proteins in the regulation of polarized divisions through their connections with the centrosomes, cytoskeleton, tissue tension and signaling pathways; and illustrates how alterations in cadherin-catenin levels or functional activity may render cells susceptible to transformation through the loss of their proliferation-differentiation balance. PMID:26451345

  9. Protein complexes and functional modules in molecular networks

    NASA Astrophysics Data System (ADS)

    Spirin, Victor; Mirny, Leonid A.

    2003-10-01

    Proteins, nucleic acids, and small molecules form a dense network of molecular interactions in a cell. Molecules are nodes of this network, and the interactions between them are edges. The architecture of molecular networks can reveal important principles of cellular organization and function, similarly to the way that protein structure tells us about the function and organization of a protein. Computational analysis of molecular networks has been primarily concerned with node degree [Wagner, A. & Fell, D. A. (2001) Proc. R. Soc. London Ser. B 268, 1803-1810; Jeong, H., Tombor, B., Albert, R., Oltvai, Z. N. & Barabasi, A. L. (2000) Nature 407, 651-654] or degree correlation [Maslov, S. & Sneppen, K. (2002) Science 296, 910-913], and hence focused on single/two-body properties of these networks. Here, by analyzing the multibody structure of the network of protein-protein interactions, we discovered molecular modules that are densely connected within themselves but sparsely connected with the rest of the network. Comparison with experimental data and functional annotation of genes showed two types of modules: (i) protein complexes (splicing machinery, transcription factors, etc.) and (ii) dynamic functional units (signaling cascades, cell-cycle regulation, etc.). Discovered modules are highly statistically significant, as is evident from comparison with random graphs, and are robust to noise in the data. Our results provide strong support for the network modularity principle introduced by Hartwell et al. [Hartwell, L. H., Hopfield, J. J., Leibler, S. & Murray, A. W. (1999) Nature 402, C47-C52], suggesting that found modules constitute the "building blocks" of molecular networks.

  10. Radiation damage to DNA-protein complexes

    NASA Astrophysics Data System (ADS)

    Spotheim-Maurizot, M.; Davídková, M.

    2011-01-01

    We review here the advances in understanding the effects of ionizing radiations on DNA, proteins and their complexes, resulting from the collaboration of the authors' teams. It concerns the preponderant indirect effect of low LET ionizing radiations, thus the attack of the macromolecules in aqueous solution by the most aggressive product of water radiolysis, the hydroxyl radical. A model of simulation of the reaction of these radicals with the macromolecules (called RADACK) was developed and was used for calculating the probabilities of damage of each constituent of DNA or proteins (nucleotide or amino-acid). The calculations allowed to draw conclusions from electrophoresis, mutagenesis, spectroscopic (fluorescence, circular dichroïsm) and mass spectrometry experiments. Thus we have shown that the extent and location of the lesions are strongly dependent on the 3D structure of the macromolecules, which in turns is modulated by their sequence and by the binding of some ligands. Molecular dynamics simulation completed our studies in showing the consequences of each lesion on the stability and structure of the proteins and their complexes with DNA.

  11. Using contrast patterns between true complexes and random subgraphs in PPI networks to predict unknown protein complexes

    PubMed Central

    Liu, Quanzhong; Song, Jiangning; Li, Jinyan

    2016-01-01

    Most protein complex detection methods utilize unsupervised techniques to cluster densely connected nodes in a protein-protein interaction (PPI) network, in spite of the fact that many true complexes are not dense subgraphs. Supervised methods have been proposed recently, but they do not answer why a group of proteins are predicted as a complex, and they have not investigated how to detect new complexes of one species by training the model on the PPI data of another species. We propose a novel supervised method to address these issues. The key idea is to discover emerging patterns (EPs), a type of contrast pattern, which can clearly distinguish true complexes from random subgraphs in a PPI network. An integrative score of EPs is defined to measure how likely a subgraph of proteins can form a complex. New complexes thus can grow from our seed proteins by iteratively updating this score. The performance of our method is tested on eight benchmark PPI datasets and compared with seven unsupervised methods, two supervised and one semi-supervised methods under five standards to assess the quality of the predicted complexes. The results show that in most cases our method achieved a better performance, sometimes significantly. PMID:26868667

  12. Using contrast patterns between true complexes and random subgraphs in PPI networks to predict unknown protein complexes.

    PubMed

    Liu, Quanzhong; Song, Jiangning; Li, Jinyan

    2016-01-01

    Most protein complex detection methods utilize unsupervised techniques to cluster densely connected nodes in a protein-protein interaction (PPI) network, in spite of the fact that many true complexes are not dense subgraphs. Supervised methods have been proposed recently, but they do not answer why a group of proteins are predicted as a complex, and they have not investigated how to detect new complexes of one species by training the model on the PPI data of another species. We propose a novel supervised method to address these issues. The key idea is to discover emerging patterns (EPs), a type of contrast pattern, which can clearly distinguish true complexes from random subgraphs in a PPI network. An integrative score of EPs is defined to measure how likely a subgraph of proteins can form a complex. New complexes thus can grow from our seed proteins by iteratively updating this score. The performance of our method is tested on eight benchmark PPI datasets and compared with seven unsupervised methods, two supervised and one semi-supervised methods under five standards to assess the quality of the predicted complexes. The results show that in most cases our method achieved a better performance, sometimes significantly.

  13. Quality Control of a Cytoplasmic Protein Complex

    PubMed Central

    Scazzari, Mario; Amm, Ingo; Wolf, Dieter H.

    2015-01-01

    For the assembly of protein complexes in the cell, the presence of stoichiometric amounts of the respective protein subunits is of utmost importance. A surplus of any of the subunits may trigger unspecific and harmful protein interactions and has to be avoided. A stoichiometric amount of subunits must finally be reached via transcriptional, translational, and/or post-translational regulation. Synthesis of saturated 16 and 18 carbon fatty acids is carried out by fatty acid synthase: in yeast Saccharomyces cerevisiae, a 2.6-MDa molecular mass assembly containing six protomers each of two different subunits, Fas1 (β) and Fas2 (α). The (α)6(β)6 complex carries six copies of all eight enzymatic activities required for fatty acid synthesis. The FAS1 and FAS2 genes in yeast are unlinked and map on two different chromosomes. Here we study the fate of the α-subunit of the complex, Fas2, when its partner, the β-subunit Fas1, is absent. Individual subunits of fatty acid synthase are proteolytically degraded when the respective partner is missing. Elimination of Fas2 is achieved by the proteasome. Here we show that a ubiquitin transfer machinery is required for Fas2 elimination. The major ubiquitin ligase targeting the superfluous Fas2 subunit to the proteasome is Ubr1. The ubiquitin-conjugating enzymes Ubc2 and Ubc4 assist the degradation process. The AAA-ATPase Cdc48 and the Hsp70 chaperone Ssa1 are crucially involved in the elimination of Fas2. PMID:25564609

  14. Joint analysis of band-specific functional connectivity and signal complexity in autism.

    PubMed

    Ghanbari, Yasser; Bloy, Luke; Christopher Edgar, J; Blaskey, Lisa; Verma, Ragini; Roberts, Timothy P L

    2015-02-01

    Examination of resting state brain activity using electrophysiological measures like complexity as well as functional connectivity is of growing interest in the study of autism spectrum disorders (ASD). The present paper jointly examined complexity and connectivity to obtain a more detailed characterization of resting state brain activity in ASD. Multi-scale entropy was computed to quantify the signal complexity, and synchronization likelihood was used to evaluate functional connectivity (FC), with node strength values providing a sensor-level measure of connectivity to facilitate comparisons with complexity. Sensor level analysis of complexity and connectivity was performed at different frequency bands computed from resting state MEG from 26 children with ASD and 22 typically developing controls (TD). Analyses revealed band-specific group differences in each measure that agreed with other functional studies in fMRI and EEG: higher complexity in TD than ASD, in frontal regions in the delta band and occipital-parietal regions in the alpha band, and lower complexity in TD than in ASD in delta (parietal regions), theta (central and temporal regions) and gamma (frontal-central boundary regions); increased short-range connectivity in ASD in the frontal lobe in the delta band and long-range connectivity in the temporal, parietal and occipital lobes in the alpha band. Finally, and perhaps most strikingly, group differences between ASD and TD in complexity and FC appear spatially complementary, such that where FC was elevated in ASD, complexity was reduced (and vice versa). The correlation of regional average complexity and connectivity node strength with symptom severity scores of ASD subjects supported the overall complementarity (with opposing sign) of connectivity and complexity measures, pointing to either diminished connectivity leading to elevated entropy due to poor inhibitory regulation or chaotic signals prohibiting effective measure of connectivity.

  15. Joint Analysis of Band-Specific Functional Connectivity and Signal Complexity in Autism

    PubMed Central

    Ghanbari, Yasser; Bloy, Luke; Edgar, J. Christopher; Blaskey, Lisa; Verma, Ragini

    2013-01-01

    Examination of resting state brain activity using electrophysiological measures like complexity as well as functional connectivity is of growing interest in the study of autism spectrum disorders (ASD). The present paper jointly examined complexity and connectivity to obtain a more detailed characterization of resting state brain activity in ASD. Multi-scale entropy was computed to quantify the signal complexity, and synchronization likelihood was used to evaluate functional connectivity (FC), with node strength values providing a sensor-level measure of connectivity to facilitate comparisons with complexity. Sensor level analysis of complexity and connectivity was performed at different frequency bands computed from resting state MEG from 26 children with ASD and 22 typically developing controls (TD). Analyses revealed band-specific group differences in each measure that agreed with other functional studies in fMRI and EEG: higher complexity in TD than ASD, in frontal regions in the delta band and occipital-parietal regions in the alpha band, and lower complexity in TD than in ASD in delta (parietal regions), theta (central and temporal regions) and gamma (frontal-central boundary regions); increased short-range connectivity in ASD in the frontal lobe in the delta band and long-range connectivity in the temporal, parietal and occipital lobes in the alpha band. Finally, and perhaps most strikingly, group differences between ASD and TD in complexity and FC appear spatially complementary, such that where FC was elevated in ASD, complexity was reduced (and vice versa). The correlation of regional average complexity and connectivity node strength with symptom severity scores of ASD subjects supported the overall complementarity (with opposing sign) of connectivity and complexity measures, pointing to either diminished connectivity leading to elevated entropy due to poor inhibitory regulation or chaotic signals prohibiting effective measure of connectivity. PMID

  16. Prioritizing protein complexes implicated in human diseases by network optimization

    PubMed Central

    2014-01-01

    Background The detection of associations between protein complexes and human inherited diseases is of great importance in understanding mechanisms of diseases. Dysfunctions of a protein complex are usually defined by its member disturbance and consequently result in certain diseases. Although individual disease proteins have been widely predicted, computational methods are still absent for systematically investigating disease-related protein complexes. Results We propose a method, MAXCOM, for the prioritization of candidate protein complexes. MAXCOM performs a maximum information flow algorithm to optimize relationships between a query disease and candidate protein complexes through a heterogeneous network that is constructed by combining protein-protein interactions and disease phenotypic similarities. Cross-validation experiments on 539 protein complexes show that MAXCOM can rank 382 (70.87%) protein complexes at the top against protein complexes constructed at random. Permutation experiments further confirm that MAXCOM is robust to the network structure and parameters involved. We further analyze protein complexes ranked among top ten for breast cancer and demonstrate that the SWI/SNF complex is potentially associated with breast cancer. Conclusions MAXCOM is an effective method for the discovery of disease-related protein complexes based on network optimization. The high performance and robustness of this approach can facilitate not only pathologic studies of diseases, but also the design of drugs targeting on multiple proteins. PMID:24565064

  17. Joint Analysis of Band-Specific Functional Connectivity and Signal Complexity in Autism

    ERIC Educational Resources Information Center

    Ghanbari, Yasser; Bloy, Luke; Edgar, J. Christopher; Blaskey, Lisa; Verma, Ragini; Roberts, Timothy P. L.

    2015-01-01

    Examination of resting state brain activity using electrophysiological measures like complexity as well as functional connectivity is of growing interest in the study of autism spectrum disorders (ASD). The present paper jointly examined complexity and connectivity to obtain a more detailed characterization of resting state brain activity in ASD.…

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

    PubMed

    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

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

    PubMed Central

    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

  20. Probing nanoparticle effect in protein-surfactant complexes

    NASA Astrophysics Data System (ADS)

    Mehan, Sumit; Aswal, V. K.; Kohlbrecher, J.

    2015-06-01

    SANS experiments have been carried to probe the role of anionic silica nanoparticles in the anionic BSA protein-cationic DTAB surfactant complexes. In protein-surfactant complex, surfactant molecules aggregate to form micelle-like clusters along the unfolded polypeptide chains of the protein. The nanoparticle aggregation mediated by oppositely charged protein-surfactant complex coexists with the free protein-surfactant complexes in the nanoparticle-protein-surfactant system. There is rearrangement of micelles in adsorbed protein-surfactant complex on nanoparticles in leading to their (nanoparticle) aggregation. On the other hand, the unfolding of protein in free protein-surfactant complex is found to be significantly enhanced in presence of nanoparticles.

  1. Immersion freezing of ice nucleation active protein complexes

    NASA Astrophysics Data System (ADS)

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

    2013-06-01

    to the outer membrane of intact bacteria or membrane fragments, (c) the temperature range in which heterogeneous droplet freezing occurs, and the fraction of droplets being able to freeze, both depend on the actual number of INA protein complexes present in the droplet ensemble, and (d) possible artifacts suspected to occur in connection with the drop freezing method, i.e., the method frequently used by biologist for quantifying ice nucleation behaviour, are of minor importance, at least for substances such as P. syringae, which induce freezing at comparably high temperatures. The last statement implies that for single ice nucleation entities such as INA protein complexes, it is the number of entities present in the droplet population, and the entities' nucleation rate, which control the freezing behaviour of the droplet population. Quantities such as ice active surface site density are not suitable in this context. The results obtained in this study allow a different perspective on the quantification of the immersion freezing behaviour of bacterial ice nucleation.

  2. The pain interactome: connecting pain-specific protein interactions.

    PubMed

    Jamieson, Daniel G; Moss, Andrew; Kennedy, Michael; Jones, Sherrie; Nenadic, Goran; Robertson, David L; Sidders, Ben

    2014-11-01

    Understanding the molecular mechanisms associated with disease is a central goal of modern medical research. As such, many thousands of experiments have been published that detail individual molecular events that contribute to a disease. Here we use a semi-automated text mining approach to accurately and exhaustively curate the primary literature for chronic pain states. In so doing, we create a comprehensive network of 1,002 contextualized protein-protein interactions (PPIs) specifically associated with pain. The PPIs form a highly interconnected and coherent structure, and the resulting network provides an alternative to those derived from connecting genes associated with pain using interactions that have not been shown to occur in a painful state. We exploit the contextual data associated with our interactions to analyse subnetworks specific to inflammatory and neuropathic pain, and to various anatomical regions. Here, we identify potential targets for further study and several drug-repurposing opportunities. Finally, the network provides a framework for the interpretation of new data within the field of pain.

  3. Engineering of complex protein sialylation in plants.

    PubMed

    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

    2016-08-23

    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

  4. Engineering of complex protein sialylation in plants

    PubMed Central

    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

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

    PubMed

    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.

  6. A New Method for Identifying Essential Proteins Based on Network Topology Properties and Protein Complexes

    PubMed Central

    Qin, Chao; Sun, Yongqi; Dong, Yadong

    2016-01-01

    Essential proteins are indispensable to the viability and reproduction of an organism. The identification of essential proteins is necessary not only for understanding the molecular mechanisms of cellular life but also for disease diagnosis, medical treatments and drug design. Many computational methods have been proposed for discovering essential proteins, but the precision of the prediction of essential proteins remains to be improved. In this paper, we propose a new method, LBCC, which is based on the combination of local density, betweenness centrality (BC) and in-degree centrality of complex (IDC). First, we introduce the common centrality measures; second, we propose the densities Den1(v) and Den2(v) of a node v to describe its local properties in the network; and finally, the combined strategy of Den1, Den2, BC and IDC is developed to improve the prediction precision. The experimental results demonstrate that LBCC outperforms traditional topological measures for predicting essential proteins, including degree centrality (DC), BC, subgraph centrality (SC), eigenvector centrality (EC), network centrality (NC), and the local average connectivity-based method (LAC). LBCC also improves the prediction precision by approximately 10 percent on the YMIPS and YMBD datasets compared to the most recently developed method, LIDC. PMID:27529423

  7. A New Method for Identifying Essential Proteins Based on Network Topology Properties and Protein Complexes.

    PubMed

    Qin, Chao; Sun, Yongqi; Dong, Yadong

    2016-01-01

    Essential proteins are indispensable to the viability and reproduction of an organism. The identification of essential proteins is necessary not only for understanding the molecular mechanisms of cellular life but also for disease diagnosis, medical treatments and drug design. Many computational methods have been proposed for discovering essential proteins, but the precision of the prediction of essential proteins remains to be improved. In this paper, we propose a new method, LBCC, which is based on the combination of local density, betweenness centrality (BC) and in-degree centrality of complex (IDC). First, we introduce the common centrality measures; second, we propose the densities Den1(v) and Den2(v) of a node v to describe its local properties in the network; and finally, the combined strategy of Den1, Den2, BC and IDC is developed to improve the prediction precision. The experimental results demonstrate that LBCC outperforms traditional topological measures for predicting essential proteins, including degree centrality (DC), BC, subgraph centrality (SC), eigenvector centrality (EC), network centrality (NC), and the local average connectivity-based method (LAC). LBCC also improves the prediction precision by approximately 10 percent on the YMIPS and YMBD datasets compared to the most recently developed method, LIDC. PMID:27529423

  8. A complex-centric view of protein network evolution.

    PubMed

    Yosef, Nir; Kupiec, Martin; Ruppin, Eytan; Sharan, Roded

    2009-07-01

    The recent availability of protein-protein interaction networks for several species makes it possible to study protein complexes in an evolutionary context. In this article, we present a novel network-based framework for reconstructing the evolutionary history of protein complexes. Our analysis is based on generalizing evolutionary measures for single proteins to the level of whole subnetworks, comprehensively considering a broad set of computationally derived complexes and accounting for both sequence and interaction changes. Specifically, we compute sets of orthologous complexes across species, and use these to derive evolutionary rate and age measures for protein complexes. We observe significant correlations between the evolutionary properties of a complex and those of its member proteins, suggesting that protein complexes form early in evolution and evolve as coherent units. Additionally, our approach enables us to directly quantify the extent to which gene duplication has played a role in the evolution of complexes. We find that about one quarter of the sets of orthologous complexes have originated from evolutionary cores of homodimers that underwent duplication and divergence, testifying to the important role of gene duplication in protein complex evolution. PMID:19465379

  9. INTERDISCIPLINARY PHYSICS AND RELATED AREAS OF SCIENCE AND TECHNOLOGY: Synchronization in Complex Networks with Multiple Connections

    NASA Astrophysics Data System (ADS)

    Wu, Qing-Chu; Fu, Xin-Chu; Sun, Wei-Gang

    2010-01-01

    In this paper a class of networks with multiple connections are discussed. The multiple connections include two different types of links between nodes in complex networks. For this new model, we give a simple generating procedure. Furthermore, we investigate dynamical synchronization behavior in a delayed two-layer network, giving corresponding theoretical analysis and numerical examples.

  10. Identification of Evening Complex Associated Proteins in Arabidopsis by Affinity Purification and Mass Spectrometry*

    PubMed Central

    Shen, Zhouxin; Kay, Steve A.

    2016-01-01

    Many species possess an endogenous circadian clock to synchronize internal physiology with an oscillating external environment. In plants, the circadian clock coordinates growth, metabolism and development over daily and seasonal time scales. Many proteins in the circadian network form oscillating complexes that temporally regulate myriad processes, including signal transduction, transcription, protein degradation and post-translational modification. In Arabidopsis thaliana, a tripartite complex composed of EARLY FLOWERING 4 (ELF4), EARLY FLOWERING 3 (ELF3), and LUX ARRHYTHMO (LUX), named the evening complex, modulates daily rhythms in gene expression and growth through transcriptional regulation. However, little is known about the physical interactions that connect the circadian system to other pathways. We used affinity purification and mass spectrometry (AP-MS) methods to identify proteins that associate with the evening complex in A. thaliana. New connections within the circadian network as well as to light signaling pathways were identified, including linkages between the evening complex, TIMING OF CAB EXPRESSION1 (TOC1), TIME FOR COFFEE (TIC), all phytochromes and TANDEM ZINC KNUCKLE/PLUS3 (TZP). Coupling genetic mutation with affinity purifications tested the roles of phytochrome B (phyB), EARLY FLOWERING 4, and EARLY FLOWERING 3 as nodes connecting the evening complex to clock and light signaling pathways. These experiments establish a hierarchical association between pathways and indicate direct and indirect interactions. Specifically, the results suggested that EARLY FLOWERING 3 and phytochrome B act as hubs connecting the clock and red light signaling pathways. Finally, we characterized a clade of associated nuclear kinases that regulate circadian rhythms, growth, and flowering in A. thaliana. Coupling mass spectrometry and genetics is a powerful method to rapidly and directly identify novel components and connections within and between complex signaling

  11. Hierarchical Organization Unveiled by Functional Connectivity in Complex Brain Networks

    NASA Astrophysics Data System (ADS)

    Zhou, Changsong; Zemanová, Lucia; Zamora, Gorka; Hilgetag, Claus C.; Kurths, Jürgen

    2006-12-01

    How do diverse dynamical patterns arise from the topology of complex networks? We study synchronization dynamics in the cortical brain network of the cat, which displays a hierarchically clustered organization, by modeling each node (cortical area) with a subnetwork of interacting excitable neurons. We find that in the biologically plausible regime the dynamics exhibits a hierarchical modular organization, in particular, revealing functional clusters coinciding with the anatomical communities at different scales. Our results provide insights into the relationship between network topology and functional organization of complex brain networks.

  12. Affinity Purification of Protein Complexes Using TAP Tags

    PubMed Central

    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

  13. The Living Career: Complexity, Chaos, Connections and Career.

    ERIC Educational Resources Information Center

    Bloch, Deborah P.

    The purpose of this paper is to present a theory of career development drawn from current work in the physical and biological sciences, specifically work that is associated with chaos and complexity theories. The paper includes specific suggestions for practice based upon the theory and reflections of career professionals on its use. The theory…

  14. The role of Hsp90 in protein complex assembly.

    PubMed

    Makhnevych, Taras; Houry, Walid A

    2012-03-01

    Hsp90 is a ubiquitous and essential molecular chaperone that plays central roles in many signaling and other cellular pathways. The in vivo and in vitro activity of Hsp90 depends on its association with a wide variety of cochaperones and cofactors, which form large multi-protein complexes involved in folding client proteins. Based on our proteomic work mapping the molecular chaperone interaction networks in yeast, especially that of Hsp90, as well as, on experiments and results presented in the published literature, one major role of Hsp90 appears to be the promotion and maintenance of proper assembly of protein complexes. To highlight this role of Hsp90, the effect of the chaperone on the assembly of the following seven complexes is discussed in this review: snoRNP, RNA polymerase II, phosphatidylinositol-3 kinase-related protein kinase (PIKK), telomere complex, kinetochore, RNA induced silencing complexes (RISC), and 26S proteasome. For some complexes, it is observed that Hsp90 mediates complex assembly by stabilizing an unstable protein subunit and facilitating its incorporation into the complex; for other complexes, Hsp90 promotes change in the composition of that complex. In all cases, Hsp90 does not appear to be part of the final assembled complex. This article is part of a Special Issue entitled:Heat Shock Protein 90 (HSP90). PMID:21945180

  15. Design and characterization of complex protein films

    NASA Astrophysics Data System (ADS)

    Bui, Holt P.

    Once a biomaterial is implanted into biological system, a layer of protein is immediately deposited on the surface of that material. The newly formed protein film will dictate how the implanted material will interact with the surrounding biological environment and lead to either the acceptance or rejection of the biomaterial. One method to enhance performance involves the activation the surface of the biomaterial with one or more proteins to direct specific interactions with the host environment. The focus of my dissertation was to develop and characterize model biomaterials surfaces that are activated with one or more proteins to help understand how the protein films may affect biological processes and a biomaterial's performance. One model system consisted of a patterned film of two proteins on a gold surface. Characterization of this protein pattern indicated that patterning protein films with a focused ion beam produced protein patterns with high biological contrast and high spatial control. The second model protein film involved the adsorption of fibronectin on surfaces with different surface energies. The characterization of the adsorbed fibronectin films suggest that fibronectin adsorbed on a hydrophilic surface is in an orientation that projects hydrophilic amino acid residues towards surface of the protein and dehydration causes reorientation to project hydrophobic amino acids towards the surface. In contrast, fibronectin is adsorbed onto a hydrophobic surface in a manner that resulted in dehydration and denaturation during the adsorption process. The last model protein film studied in this work consisted of fibronectin patterned in a manner so that the film consisted of spatially controlled domains of fibronectin adsorbed onto a hydrophilic surface as well as a hydrophobic surface. Lateral characterization of this pattern demonstrated a difference in secondary structure of fibronectin adsorbed on the two domains with varying surface energies.

  16. Conservation and variability of synaptonemal complex proteins in phylogenesis of eukaryotes.

    PubMed

    Grishaeva, Tatiana M; Bogdanov, Yuri F

    2014-01-01

    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. PMID:25147749

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

    PubMed Central

    Grishaeva, Tatiana M.; Bogdanov, Yuri F.

    2014-01-01

    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. PMID:25147749

  18. Conservation and variability of synaptonemal complex proteins in phylogenesis of eukaryotes.

    PubMed

    Grishaeva, Tatiana M; Bogdanov, Yuri F

    2014-01-01

    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.

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

    PubMed

    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.

    PubMed

    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.

    PubMed

    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. Multi-LZerD: Multiple protein docking for asymmetric complexes

    PubMed Central

    Esquivel-Rodríguez, Juan; Yang, Yifeng David; Kihara, Daisuke

    2012-01-01

    The tertiary structures of protein complexes provide a crucial insight about the molecular mechanisms that regulate their functions and assembly. However, solving protein complex structures by experimental methods is often more difficult than single protein structures. Here, we have developed a novel computational multiple protein docking algorithm, Multi-LZerD, that builds models of multimeric complexes by effectively reusing pairwise docking predictions of component proteins. A genetic algorithm is applied to explore the conformational space followed by a structure refinement procedure. Benchmark on eleven hetero-multimeric complexes resulted in near native conformations for all but one of them (a root mean square deviation smaller than 2.5Å). We also show that our method copes with unbound docking cases well, outperforming the methodology that can be directly compared to our approach. Multi-LZerD was able to predict near native structures for multimeric complexes of various topologies. PMID:22488467

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

    PubMed

    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.

  4. Studying protein complexes by the yeast two-hybrid system.

    PubMed

    Rajagopala, Seesandra V; Sikorski, Patricia; Caufield, J Harry; Tovchigrechko, Andrey; Uetz, Peter

    2012-12-01

    Protein complexes are typically analyzed by affinity purification and subsequent mass spectrometric analysis. However, in most cases the structure and topology of the complexes remains elusive from such studies. Here we investigate how the yeast two-hybrid system can be used to analyze direct interactions among proteins in a complex. First we tested all pairwise interactions among the seven proteins of Escherichia coli DNA polymerase III as well as an uncharacterized complex that includes MntR and PerR. Four and seven interactions were identified in these two complexes, respectively. In addition, we review Y2H data for three other complexes of known structure which serve as "gold-standards", namely Varicella Zoster Virus (VZV) ribonucleotide reductase (RNR), the yeast proteasome, and bacteriophage lambda. Finally, we review an Y2H analysis of the human spliceosome which may serve as an example for a dynamic mega-complex.

  5. Immersion freezing of ice nucleating active protein complexes

    NASA Astrophysics Data System (ADS)

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

    2012-08-01

    Biological particles, e.g. bacteria and their Ice Nucleating Active (INA) protein complexes, might play an important role for the ice formation in atmospheric mixed-phase clouds. Therefore, the immersion freezing behavior of INA protein complexes generated from a SnomaxTM solution/suspension was investigated as function of temperature in a range of -5 °C to -38 °C at the Leipzig Aerosol Cloud Interaction Simulator (LACIS). The immersion freezing of droplets containing small numbers of INA protein complexes occurs in a temperature range of -7 °C and -10 °C. The experiments performed in the lower temperature range, where all droplets freeze which contain at least one INA protein complex, are used to determine the average number of INA protein complexes present, assuming that the INA protein complexes are Poisson distributed over the droplet ensemble. Knowing the average number of INA protein complexes, the heterogeneous ice nucleation rate and rate coefficient of a single INA protein complex is determined by using the newly-developed CHESS model (stoCHastic model of idEntical poiSSon distributed ice nuclei). Therefore, we assume the ice nucleation process to be of stochastic nature, and a parameterization of the INA protein complex's nucleation rate. Analyzing the results of immersion freezing experiments from literature (SnomaxTM and Pseudomonas syringae bacteria), to results gained in this study, demonstrates that first, a similar temperature dependence of the heterogeneous ice nucleation rate for a single INA protein complex was found in all experiments, second, the shift of the ice fraction curves to higher temperatures can be explained consistently by a higher average number of INA protein complexes being present in the droplet ensemble, and finally the heterogeneous ice nucleation rate of one single INA protein complex might be also applicable for intact Pseudomonas syringae bacteria cells. The results obtained in this study allow a new perspective on the

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

    PubMed

    Lindhoud, Saskia; Claessens, Mireille M A E

    2016-01-14

    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 function of the mixing ratio, protein concentration and ionic strength. Maximal protein enrichment of the complex coacervate phase was observed to require the presence of all three macromolecules. Under optimized conditions the protein concentrations in the complex coacervate were as high as 200 g L(-1). Such high concentrations are comparable to the protein concentration in the cytosol, suggesting that these interesting liquid phases may serve a suitable model system for the phase behavior of the cytosol and genesis and function of membrane-less organelles. The high stability of the complexes and the salt dependent uptake of protein suggest that complex coacervates may provide a way to store hydrated proteins at high concentrations and might therefore be of interest in the formulation of high protein foods.

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

    PubMed Central

    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

  8. The responsive amygdala: treatment-induced alterations in functional connectivity in pediatric complex regional pain syndrome.

    PubMed

    Simons, L E; Pielech, M; Erpelding, N; Linnman, C; Moulton, E; Sava, S; Lebel, A; Serrano, P; Sethna, N; Berde, C; Becerra, L; Borsook, D

    2014-09-01

    The amygdala is a key brain region with efferent and afferent neural connections that involve complex behaviors such as pain, reward, fear, and anxiety. This study evaluated resting state functional connectivity of the amygdala with cortical and subcortical regions in a group of chronic pain patients (pediatric complex regional pain syndrome) with age-sex matched control subjects before and after intensive physical-biobehavioral pain treatment. Our main findings include (1) enhanced functional connectivity from the amygdala to multiple cortical, subcortical, and cerebellar regions in patients compared with control subjects, with differences predominantly in the left amygdala in the pretreated condition (disease state); (2) dampened hyperconnectivity from the left amygdala to the motor cortex, parietal lobe, and cingulate cortex after intensive pain rehabilitation treatment within patients with nominal differences observed among healthy control subjects from time 1 to time 2 (treatment effects); (3) functional connectivity to several regions key to fear circuitry (prefrontal cortex, bilateral middle temporal lobe, bilateral cingulate, hippocampus) correlated with higher pain-related fear scores; and (4) decreases in pain-related fear associated with decreased connectivity between the amygdala and the motor and somatosensory cortex, cingulate, and frontal areas. Our data suggest that there are rapid changes in amygdala connectivity after an aggressive treatment program in children with chronic pain and intrinsic amygdala functional connectivity activity serving as a potential indicator of treatment response.

  9. Mass Spectrometry of Protein Complexes: From Origins to Applications

    NASA Astrophysics Data System (ADS)

    Mehmood, Shahid; Allison, Timothy M.; Robinson, Carol V.

    2015-04-01

    Now routine is the ability to investigate soluble and membrane protein complexes in the gas phase of a mass spectrometer while preserving folded structure and ligand-binding properties. Several recent transformative developments have occurred to arrive at this point. These include advances in mass spectrometry instrumentation, particularly with respect to resolution; the ability to study intact membrane protein complexes released from detergent micelles; and the use of protein unfolding in the gas phase to obtain stability parameters. Together, these discoveries are providing unprecedented information on the compositional heterogeneity of biomacromolecules, the unfolding trajectories of multidomain proteins, and the stability imparted by ligand binding to both soluble and membrane-embedded protein complexes. We review these recent breakthroughs, highlighting the challenges that had to be overcome and the physicochemical insight that can now be gained from studying proteins and their assemblies in the gas phase.

  10. Large, dynamic, multi-protein complexes: a challenge for structural biology.

    PubMed

    Różycki, Bartosz; Boura, Evzen

    2014-11-19

    Structural biology elucidates atomic structures of macromolecules such as proteins, DNA, RNA, and their complexes to understand the basic mechanisms of their functions. Among proteins that pose the most difficult problems to current efforts are those which have several large domains connected by long, flexible polypeptide segments. Although abundant and critically important in biological cells, such proteins have proven intractable by conventional techniques. This gap has recently led to the advancement of hybrid methods that use state-of-the-art computational tools to combine complementary data from various high- and low-resolution experiments. In this review, we briefly discuss the individual experimental techniques to illustrate their strengths and limitations, and then focus on the use of hybrid methods in structural biology. We describe how representative structures of dynamic multi-protein complexes are obtained utilizing the EROS hybrid method that we have co-developed. PMID:25335513

  11. Large, dynamic, multi-protein complexes: a challenge for structural biology

    NASA Astrophysics Data System (ADS)

    Różycki, Bartosz; Boura, Evzen

    2014-11-01

    Structural biology elucidates atomic structures of macromolecules such as proteins, DNA, RNA, and their complexes to understand the basic mechanisms of their functions. Among proteins that pose the most difficult problems to current efforts are those which have several large domains connected by long, flexible polypeptide segments. Although abundant and critically important in biological cells, such proteins have proven intractable by conventional techniques. This gap has recently led to the advancement of hybrid methods that use state-of-the-art computational tools to combine complementary data from various high- and low-resolution experiments. In this review, we briefly discuss the individual experimental techniques to illustrate their strengths and limitations, and then focus on the use of hybrid methods in structural biology. We describe how representative structures of dynamic multi-protein complexes are obtained utilizing the EROS hybrid method that we have co-developed.

  12. Protein camouflage in cytochrome c-calixarene complexes

    NASA Astrophysics Data System (ADS)

    McGovern, Róise E.; Fernandes, Humberto; Khan, Amir R.; Power, Nicholas P.; Crowley, Peter B.

    2012-07-01

    Small molecules that recognize protein surfaces are important tools for modifying protein interaction properties. Since the 1980s, several thousand studies concerning calixarenes and host-guest interactions have been published. Although there is growing interest in protein-calixarene interactions, only limited structural information has been available to date. We now report the crystal structure of a protein-calixarene complex. The water-soluble p-sulfonatocalix[4]arene is shown to bind the lysine-rich cytochrome c at three different sites. Binding curves obtained from NMR titrations reveal an interaction process that involves two or more binding sites. Together, the data indicate a dynamic complex in which the calixarene explores the surface of cytochrome c. In addition to providing valuable information on protein recognition, the data also indicate that the calixarene is a mediator of protein-protein interactions, with potential applications in generating assemblies and promoting crystallization.

  13. Connectivity in the human brain dissociates entropy and complexity of auditory inputs.

    PubMed

    Nastase, Samuel A; Iacovella, Vittorio; Davis, Ben; Hasson, Uri

    2015-03-01

    Complex systems are described according to two central dimensions: (a) the randomness of their output, quantified via entropy; and (b) their complexity, which reflects the organization of a system's generators. Whereas some approaches hold that complexity can be reduced to uncertainty or entropy, an axiom of complexity science is that signals with very high or very low entropy are generated by relatively non-complex systems, while complex systems typically generate outputs with entropy peaking between these two extremes. In understanding their environment, individuals would benefit from coding for both input entropy and complexity; entropy indexes uncertainty and can inform probabilistic coding strategies, whereas complexity reflects a concise and abstract representation of the underlying environmental configuration, which can serve independent purposes, e.g., as a template for generalization and rapid comparisons between environments. Using functional neuroimaging, we demonstrate that, in response to passively processed auditory inputs, functional integration patterns in the human brain track both the entropy and complexity of the auditory signal. Connectivity between several brain regions scaled monotonically with input entropy, suggesting sensitivity to uncertainty, whereas connectivity between other regions tracked entropy in a convex manner consistent with sensitivity to input complexity. These findings suggest that the human brain simultaneously tracks the uncertainty of sensory data and effectively models their environmental generators.

  14. Embracing proteins: structural themes in aptamer-protein complexes.

    PubMed

    Gelinas, Amy D; Davies, Douglas R; Janjic, Nebojsa

    2016-02-01

    Understanding the structural rules that govern specific, high-affinity binding characteristic of aptamer-protein interactions is important in view of the increasing use of aptamers across many applications. From the modest number of 16 aptamer-protein structures currently available, trends are emerging. The flexible phosphodiester backbone allows folding into precise three-dimensional structures using known nucleic acid motifs as scaffolds that orient specific functional groups for target recognition. Still, completely novel motifs essential for structure and function are found in modified aptamers with diversity-enhancing side chains. Aptamers and antibodies, two classes of macromolecules used as affinity reagents with entirely different backbones and composition, recognize protein epitopes of similar size and with comparably high shape complementarity. PMID:26919170

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

    PubMed

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

    2012-12-01

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

  16. Cell density signal protein suitable for treatment of connective tissue injuries and defects

    DOEpatents

    Schwarz, Richard I.

    2002-08-13

    Identification, isolation and partial sequencing of a cell density protein produced by fibroblastic cells. The cell density signal protein comprising a 14 amino acid peptide or a fragment, variant, mutant or analog thereof, the deduced cDNA sequence from the 14 amino acid peptide, a recombinant protein, protein and peptide-specific antibodies, and the use of the peptide and peptide-specific antibodies as therapeutic agents for regulation of cell differentiation and proliferation. A method for treatment and repair of connective tissue and tendon injuries, collagen deficiency, and connective tissue defects.

  17. Structural study of coacervation in protein-polyelectrolyte complexes

    NASA Astrophysics Data System (ADS)

    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

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

    PubMed

    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.

  19. Identification of Post-translational Modifications of Plant Protein Complexes

    PubMed Central

    Piquerez, Sophie J. M.; Balmuth, Alexi L.; Sklenář, Jan; Jones, Alexandra M.E.; Rathjen, John P.; Ntoukakis, Vardis

    2014-01-01

    Plants adapt quickly to changing environments due to elaborate perception and signaling systems. During pathogen attack, plants rapidly respond to infection via the recruitment and activation of immune complexes. Activation of immune complexes is associated with post-translational modifications (PTMs) of proteins, such as phosphorylation, glycosylation, or ubiquitination. Understanding how these PTMs are choreographed will lead to a better understanding of how resistance is achieved. Here we describe a protein purification method for nucleotide-binding leucine-rich repeat (NB-LRR)-interacting proteins and the subsequent identification of their post-translational modifications (PTMs). With small modifications, the protocol can be applied for the purification of other plant protein complexes. The method is based on the expression of an epitope-tagged version of the protein of interest, which is subsequently partially purified by immunoprecipitation and subjected to mass spectrometry for identification of interacting proteins and PTMs. This protocol demonstrates that: i). Dynamic changes in PTMs such as phosphorylation can be detected by mass spectrometry; ii). It is important to have sufficient quantities of the protein of interest, and this can compensate for the lack of purity of the immunoprecipitate; iii). In order to detect PTMs of a protein of interest, this protein has to be immunoprecipitated to get a sufficient quantity of protein. PMID:24637539

  20. Coupling protein complex analysis to peptide based proteomics.

    PubMed

    Gao, Qiang; Madian, Ashraf G; Liu, Xiuping; Adamec, Jiri; Regnier, Fred E

    2010-12-01

    Proteolysis is a central component of most proteomics methods. Unfortunately much of the information relating to the structural diversity of proteins is lost during digestion. This paper describes a method in which the native proteome of yeast was subjected to preliminary fractionation by size exclusion chromatography (SEC) prior to trypsin digestion of SEC fractions and reversed phase chromatography-mass spectral analysis to identify tryptic peptides thus generated. Through this approach proteins associated with other proteins in high molecular mass complexes were recognized and identified. A focus of this work was on the identification of Hub proteins that associate with multiple interaction partners. A critical component of this strategy is to choose methods and conditions that maximize retention of native structure during the various stages of analysis prior to proteolysis, especially during cell lysis. Maximum survival of protein complexes during lysis was obtained with the French press and bead-beater methods of cell disruption at approximately pH 8 with 200 mM NaCl in the lysis buffer. Structure retention was favored by higher ionic strength, suggesting that hydrophobic effects are important in maintaining the structure of protein complexes. Recovery of protein complexes declined substantially with storage at any temperature, but storage at -20°C was best when low temperature storage was necessary. Slightly lower recovery was obtained with storage at -80°C while lowest recovery was achieved at 4°C. It was concluded that initial fractionation of native proteins in cell lysates by SEC prior to RPC-MS/MS of tryptic digests can be used to recognize and identify proteins in complexes along with their interaction partners in known protein complexes.

  1. Connectivity

    ERIC Educational Resources Information Center

    Grush, Mary, Ed.

    2006-01-01

    Connectivity has dramatically changed the landscape of higher education IT. From "on-demand" services for net-gen students and advanced eLearning systems for faculty, to high-performance computing grid resources for researchers, IT now provides more networked services than ever to connect campus constituents to each other and to the world.…

  2. Visualizing active membrane protein complexes by electron cryotomography

    PubMed Central

    Gold, Vicki A.M.; Ieva, Raffaele; Walter, Andreas; Pfanner, Nikolaus; van der Laan, Martin; Kühlbrandt, Werner

    2014-01-01

    Unravelling the structural organization of membrane protein machines in their active state and native lipid environment is a major challenge in modern cell biology research. Here we develop the STAMP (Specifically TArgeted Membrane nanoParticle) technique as a strategy to localize protein complexes in situ by electron cryotomography (cryo-ET). STAMP selects active membrane protein complexes and marks them with quantum dots. Taking advantage of new electron detector technology that is currently revolutionizing cryotomography in terms of achievable resolution, this approach enables us to visualize the three-dimensional distribution and organization of protein import sites in mitochondria. We show that import sites cluster together in the vicinity of crista membranes, and we reveal unique details of the mitochondrial protein import machinery in action. STAMP can be used as a tool for site-specific labelling of a multitude of membrane proteins by cryo-ET in the future. PMID:24942077

  3. Developing and Modeling Complex Social Interventions: Introducing the Connecting People Intervention

    ERIC Educational Resources Information Center

    Webber, Martin; Reidy, Hannah; Ansari, David; Stevens, Martin; Morris, David

    2016-01-01

    Objectives: Modeling the processes involved in complex social interventions is important in social work practice, as it facilitates their implementation and translation into different contexts. This article reports the process of developing and modeling the connecting people intervention (CPI), a model of practice that supports people with mental…

  4. Biochemical isolation of Argonaute protein complexes by Ago-APP

    PubMed Central

    Hauptmann, Judith; Schraivogel, Daniel; Bruckmann, Astrid; Manickavel, Sudhir; Jakob, Leonhard; Eichner, Norbert; Pfaff, Janina; Urban, Marc; Sprunck, Stefanie; Hafner, Markus; Tuschl, Thomas; Deutzmann, Rainer; Meister, Gunter

    2015-01-01

    During microRNA (miRNA)-guided gene silencing, Argonaute (Ago) proteins interact with a member of the TNRC6/GW protein family. Here we used a short GW protein-derived peptide fused to GST and demonstrate that it binds to Ago proteins with high affinity. This allows for the simultaneous isolation of all Ago protein complexes expressed in diverse species to identify associated proteins, small RNAs, or target mRNAs. We refer to our method as “Ago protein Affinity Purification by Peptides“ (Ago-APP). Furthermore, expression of this peptide competes for endogenous TNRC6 proteins, leading to global inhibition of miRNA function in mammalian cells. PMID:26351695

  5. The tandem affinity purification method: an efficient system for protein complex purification and protein interaction identification.

    PubMed

    Xu, Xiaoli; Song, Yuan; Li, Yuhua; Chang, Jianfeng; Zhang, Hua; An, Lizhe

    2010-08-01

    Isolation and identification of protein partners in multi-protein complexes are important in gaining further insights into the cellular roles of proteins and determining the possible mechanisms by which proteins have an effect in the molecular environment. The tandem affinity purification (TAP) method was originally developed in yeast for the purification of protein complexes and identification of protein-protein interactions. With modifications to this method and many variations in the original tag made over the past few years, the TAP system could be applied in mammalian, plant, bacteria and other systems for protein complex analysis. In this review, we describe the application of the TAP method in various organisms, the modification in the tag, the disadvantages, the developments and the future prospects of the TAP method. PMID:20399864

  6. A Diatom Light-Harvesting Pigment-Protein Complex 1

    PubMed Central

    Friedman, Alan L.; Alberte, Randall S.

    1984-01-01

    A light-harvesting pigment-protein complex was isolated from the diatom Phaeodactylum tricornutum using the zwitterionic detergent CHAPS (3-[3-cholamidopropyl)dimethylammonio]-1-propanesulfonate). Detergent-solubilized membranes were fractionated by sucrose density gradient centrifugation into three components. The medium density fraction contained chlorophyll a, chlorophyll c, and fucoxanthin. This fraction was purified by DEAE-ion exchange chromatography, and contained chlorophyll a, chlorophyll c, and fucoxanthin in a molar ratio of 2.4:1.0:4.8. Fluorescence emission and excitation spectra of the isolated complex demonstrated that light energy absorbed by chlorophyll c and fucoxanthin was coupled to chlorophyll a fluorescence. Upon denaturation, the apoprotein yielded a polypeptide doublet at 17.5 to 18.0 kilodaltons which accounted for 30 to 40% of the toal membrane protein. These findings indicate that this pigment-protein complex is a major component of the diatom photosynthetic lammellae. The quantitative amino acid composition of the apoprotein was very similar to those reported for other membrane-bound pigment-protein complexes. Based on the protein to chlorophyll a ratio of 7700 grams protein per mole chlorophyll a for the complex, each apoprotein molecule contains, to the nearest integer, two chlorophyll a, one chlorophyll c, and five fucoxanthin molecules. Polyclonal antibodies raised against the 17.5 to 18.0 kilodaltons apoprotein showed a monospecific reaction with only the 17.5 to 18.0 protein zone from denatured P. tricornutum membranes as well as to the nondenatured pigment-protein complex. It appears that this complex is common to other diatom species. Images Fig. 2 Fig. 3 PMID:16663869

  7. A local average connectivity-based method for identifying essential proteins from the network level.

    PubMed

    Li, Min; Wang, Jianxin; Chen, Xiang; Wang, Huan; Pan, Yi

    2011-06-01

    Identifying essential proteins is very important for understanding the minimal requirements of cellular survival and development. Fast growth in the amount of available protein-protein interactions has produced unprecedented opportunities for detecting protein essentiality from the network level. Essential proteins have been found to be more abundant among those highly connected proteins. However, there exist a number of highly connected proteins which are not essential. By analyzing these proteins, we find that few of their neighbors interact with each other. Thus, we propose a new local method, named LAC, to determine a protein's essentiality by evaluating the relationship between a protein and its neighbors. The performance of LAC is validated based on the yeast protein interaction networks obtained from two different databases: DIP and BioGRID. The experimental results of the two networks show that the number of essential proteins predicted by LAC clearly exceeds that explored by Degree Centrality (DC). More over, LAC is also compared with other seven measures of protein centrality (Neighborhood Component (DMNC), Betweenness Centrality (BC), Closeness Centrality (CC), Bottle Neck (BN), Information Centrality (IC), Eigenvector Centrality (EC), and Subgraph Centrality (SC)) in identifying essential proteins. The comparison results based on the validations of sensitivity, specificity, F-measure, positive predictive value, negative predictive value, and accuracy consistently show that LAC outweighs these seven previous methods. PMID:21704260

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

    PubMed

    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.

  9. Protein Complex Production from the Drug Discovery Standpoint.

    PubMed

    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.

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

    PubMed Central

    Luo, Jiawei; Qi, Yi

    2015-01-01

    Background 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. Method 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. Results 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). Conclusions LIDC is more effective for the prediction of essential proteins than other recently developed methods. PMID:26125187

  11. Transcriptional regulation of protein complexes within and across species.

    PubMed

    Tan, Kai; Shlomi, Tomer; Feizi, Hoda; Ideker, Trey; Sharan, Roded

    2007-01-23

    Yeast two-hybrid and coimmunoprecipitation experiments have defined large-scale protein-protein interaction networks for many model species. Separately, systematic chromatin immunoprecipitation experiments have enabled the assembly of large networks of transcriptional regulatory interactions. To investigate the functional interplay between these two interaction types, we combined both within a probabilistic framework that models the cell as a network of transcription factors regulating protein complexes. This framework identified 72 putative coregulated complexes in yeast and allowed the prediction of 120 previously uncharacterized transcriptional interactions. Several predictions were tested by new microarray profiles, yielding a confirmation rate (58%) comparable with that of direct immunoprecipitation experiments. Furthermore, we extended our framework to a cross-species setting, identifying 24 coregulated complexes that were conserved between yeast and fly. Analyses of these conserved complexes revealed different conservation levels of their regulators and provided suggestive evidence that protein-protein interaction networks may evolve more slowly than transcriptional interaction networks. Our results demonstrate how multiple molecular interaction types can be integrated toward a global wiring diagram of the cell, and they provide insights into the evolutionary dynamics of protein complex regulation.

  12. Proteins associated with RNase E in a multicomponent ribonucleolytic complex.

    PubMed Central

    Miczak, A; Kaberdin, V R; Wei, C L; Lin-Chao, S

    1996-01-01

    The Escherichia coli endoribonuclease RNase E is essential for RNA processing and degradation. Earlier work provided evidence that RNase E exists intracellularly as part of a multicomponent complex and that one of the components of this complex is a 3'-to-5' exoribonuclease, polynucleotide phosphorylase (EC 2.7.7.8). To isolate and identify other components of the RNase E complex, FLAG-epitope-tagged RNase E (FLAG-Rne) fusion protein was purified on a monoclonal antibody-conjugated agarose column. The FLAG-Rne fusion protein, eluted by competition with the synthetic FLAG peptide, was found to be associated with other proteins. N-terminal sequencing of these proteins revealed the presence in the RNase E complex not only of polynucleotide phosphorylase but also of DnaK, RNA helicase, and enolase (EC 4.2.1.11). Another protein associated only with epitope-tagged temperature-sensitive (Rne-3071) mutant RNase E but not with the wild-type enzyme is GroEL. The FLAG-Rne complex has RNase E activity in vivo and in vitro. The relative amount of proteins associated with wild-type and Rne-3071 expressed at an elevated temperature differed. Images Fig. 1 Fig. 2 PMID:8632981

  13. Connecting Protein Structure to Intermolecular Interactions: A Computer Modeling Laboratory

    ERIC Educational Resources Information Center

    Abualia, Mohammed; Schroeder, Lianne; Garcia, Megan; Daubenmire, Patrick L.; Wink, Donald J.; Clark, Ginevra A.

    2016-01-01

    An understanding of protein folding relies on a solid foundation of a number of critical chemical concepts, such as molecular structure, intra-/intermolecular interactions, and relating structure to function. Recent reports show that students struggle on all levels to achieve these understandings and use them in meaningful ways. Further, several…

  14. Negative Ions Enhance Survival of Membrane Protein Complexes

    NASA Astrophysics Data System (ADS)

    Liko, Idlir; Hopper, Jonathan T. S.; Allison, Timothy M.; Benesch, Justin L. P.; Robinson, Carol V.

    2016-06-01

    Membrane protein complexes are commonly introduced to the mass spectrometer solubilized in detergent micelles. The collisional activation used to remove the detergent, however, often causes protein unfolding and dissociation. As in the case for soluble proteins, electrospray in the positive ion mode is most commonly used for the study of membrane proteins. Here we show several distinct advantages of employing the negative ion mode. Negative polarity can yield lower average charge states for membrane proteins solubilized in saccharide detergents, with enhanced peak resolution and reduced adduct formation. Most importantly, we demonstrate that negative ion mode electrospray ionization (ESI) minimizes subunit dissociation in the gas phase, allowing access to biologically relevant oligomeric states. Together, these properties mean that intact membrane protein ions can be generated in a greater range of solubilizing detergents. The formation of negative ions, therefore, greatly expands the possibilities of using mass spectrometry on this intractable class of protein.

  15. Fish assemblages, connectivity, and habitat rehabilitation in a diked Great Lakes coastal wetland complex

    USGS Publications Warehouse

    Kowalski, Kurt P.; Wiley, Michael J.; Wilcox, Douglas A.

    2014-01-01

    Fish and plant assemblages in the highly modified Crane Creek coastal wetland complex of Lake Erie were sampled to characterize their spatial and seasonal patterns and to examine the implications of the hydrologic connection of diked wetland units to Lake Erie. Fyke netting captured 52 species and an abundance of fish in the Lake Erie–connected wetlands, but fewer than half of those species and much lower numbers and total masses of fish were captured in diked wetland units. Although all wetland units were immediately adjacent to Lake Erie, there were also pronounced differences in water quality and wetland vegetation between the hydrologically isolated and lake-connected wetlands. Large seasonal variations in fish assemblage composition and biomass were observed in connected wetland units but not in disconnected units. Reestablishment of hydrologic connectivity in diked wetland units would allow coastal Lake Erie fish to use these vegetated habitats seasonally, although connectivity does appear to pose some risks, such as the expansion of invasive plants and localized reductions in water quality. Periodic isolation and drawdown of the diked units could still be used to mimic intermediate levels of disturbance and manage invasive wetland vegetation.

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

    PubMed Central

    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

  17. Subunit connectivity, assembly determinants, and architecture of the yeast exocyst complex

    PubMed Central

    Heider, Margaret R.; Gu, Mingyu; Duffy, Caroline M.; Mirza, Anne M.; Marcotte, Laura L.; Walls, Alexandra C.; Farrall, Nicholas; Hakhverdyan, Zhanna; Field, Mark C.; Rout, Michael P.; Frost, Adam; Munson, Mary

    2016-01-01

    The exocyst is a hetero-octameric complex proposed to serve as the tethering complex for exocytosis, although it remains poorly understood at the molecular level. Here, we purified endogenous exocyst from Saccharomyces cerevisiae, and show that the purified complexes are stable and consist of all eight subunits with equal stoichiometry. Using a combination of biochemical and auxin-induced degradation experiments in yeast, we mapped the subunit connectivity, identified two stable four-subunit modules within the octamer, and demonstrated that several known exocyst binding partners are not necessary for exocyst assembly and stability. Furthermore, we visualized the structure of the yeast complex using negative stain electron microscopy; our results indicate that exocyst exists predominantly as a stable, octameric complex with an elongated architecture that suggests the subunits are contiguous helical bundles packed together into a bundle of long rods. PMID:26656853

  18. Chimeric Protein Complexes in Hybrid Species Generate Novel Phenotypes

    PubMed Central

    Piatkowska, Elzbieta M.; Naseeb, Samina; Knight, David; Delneri, Daniela

    2013-01-01

    Hybridization between species is an important mechanism for the origin of novel lineages and adaptation to new environments. Increased allelic variation and modification of the transcriptional network are the two recognized forces currently deemed to be responsible for the phenotypic properties seen in hybrids. However, since the majority of the biological functions in a cell are carried out by protein complexes, inter-specific protein assemblies therefore represent another important source of natural variation upon which evolutionary forces can act. Here we studied the composition of six protein complexes in two different Saccharomyces “sensu stricto” hybrids, to understand whether chimeric interactions can be freely formed in the cell in spite of species-specific co-evolutionary forces, and whether the different types of complexes cause a change in hybrid fitness. The protein assemblies were isolated from the hybrids via affinity chromatography and identified via mass spectrometry. We found evidence of spontaneous chimericity for four of the six protein assemblies tested and we showed that different types of complexes can cause a variety of phenotypes in selected environments. In the case of TRP2/TRP3 complex, the effect of such chimeric formation resulted in the fitness advantage of the hybrid in an environment lacking tryptophan, while only one type of parental combination of the MBF complex allowed the hybrid to grow under respiratory conditions. These phenotypes were dependent on both genetic and environmental backgrounds. This study provides empirical evidence that chimeric protein complexes can freely assemble in cells and reveals a new mechanism to generate phenotypic novelty and plasticity in hybrids to complement the genomic innovation resulting from gene duplication. The ability to exchange orthologous members has also important implications for the adaptation and subsequent genome evolution of the hybrids in terms of pattern of gene loss. PMID

  19. Reconstitution of multivalent PDZ domain binding to the scaffold protein PSD-95 reveals ternary-complex specificity of combinatorial inhibition

    PubMed Central

    McCann, James J.; Choi, Ucheor B.; Bowen, Mark E.

    2014-01-01

    Summary Multi-domain scaffold proteins serve as hubs in the signal transduction network. By physically colocalizing sequential steps in a transduction pathway, scaffolds catalyze and direct incoming signals. Much is known about binary interactions with individual domains but it is unknown whether “scaffolding activity” is predictable from pairwise affinities. Here, we characterized multivalent binding to PSD-95, a scaffold protein containing three PDZ domains connected in series by disordered linkers. We used single molecule fluorescence to watch soluble PSD-95 recruit diffusing proteins to a surface-attached receptor cytoplasmic domain. Different ternary complexes showed unique concentration dependence for scaffolding despite similar pairwise affinity. The concentration dependence of scaffolding activity was not predictable based on binary interactions. PSD-95 did not stabilize specific complexes, but rather increased the frequency of transient binding events. Our results suggest that PSD-95 maintains a loosely-connected pleomorphic ensemble rather than forming a stereospecific complex containing all components. PMID:25220472

  20. Emergence of Complexity in Protein Functions and Metabolic Networks

    NASA Technical Reports Server (NTRS)

    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

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

    PubMed

    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.).

  2. Connecting two proteins using a fusion alpha helix stabilized by a chemical cross linker

    NASA Astrophysics Data System (ADS)

    Jeong, Woo Hyeon; Lee, Haerim; Song, Dong Hyun; Eom, Jae-Hoon; Kim, Sun Chang; Lee, Hee-Seung; Lee, Hayyoung; Lee, Jie-Oh

    2016-03-01

    Building a sophisticated protein nano-assembly requires a method for linking protein components in a predictable and stable structure. Most of the cross linkers available have flexible spacers. Because of this, the linked hybrids have significant structural flexibility and the relative structure between their two components is largely unpredictable. Here we describe a method of connecting two proteins via a `fusion α helix' formed by joining two pre-existing helices into a single extended helix. Because simple ligation of two helices does not guarantee the formation of a continuous helix, we used EY-CBS, a synthetic cross linker that has been shown to react selectively with cysteines in α-helices, to stabilize the connecting helix. Formation and stabilization of the fusion helix was confirmed by determining the crystal structures of the fusion proteins with and without bound EY-CBS. Our method should be widely applicable for linking protein building blocks to generate predictable structures.

  3. Connecting two proteins using a fusion alpha helix stabilized by a chemical cross linker

    PubMed Central

    Jeong, Woo Hyeon; Lee, Haerim; Song, Dong Hyun; Eom, Jae-Hoon; Kim, Sun Chang; Lee, Hee-Seung; Lee, Hayyoung; Lee, Jie-Oh

    2016-01-01

    Building a sophisticated protein nano-assembly requires a method for linking protein components in a predictable and stable structure. Most of the cross linkers available have flexible spacers. Because of this, the linked hybrids have significant structural flexibility and the relative structure between their two components is largely unpredictable. Here we describe a method of connecting two proteins via a ‘fusion α helix' formed by joining two pre-existing helices into a single extended helix. Because simple ligation of two helices does not guarantee the formation of a continuous helix, we used EY-CBS, a synthetic cross linker that has been shown to react selectively with cysteines in α-helices, to stabilize the connecting helix. Formation and stabilization of the fusion helix was confirmed by determining the crystal structures of the fusion proteins with and without bound EY-CBS. Our method should be widely applicable for linking protein building blocks to generate predictable structures. PMID:26980593

  4. Synthetic RNA-protein complex shaped like an equilateral triangle

    NASA Astrophysics Data System (ADS)

    Ohno, Hirohisa; Kobayashi, Tetsuhiro; Kabata, Rinko; Endo, Kei; Iwasa, Takuma; Yoshimura, Shige H.; Takeyasu, Kunio; Inoue, Tan; Saito, Hirohide

    2011-02-01

    Synthetic nanostructures consisting of biomacromolecules such as nucleic acids have been constructed using bottom-up approaches. In particular, Watson-Crick base pairing has been used to construct a variety of two- and three-dimensional DNA nanostructures. Here, we show that RNA and the ribosomal protein L7Ae can form a nanostructure shaped like an equilateral triangle that consists of three proteins bound to an RNA scaffold. The construction of the complex relies on the proteins binding to kink-turn (K-turn) motifs in the RNA, which allows the RNA to bend by ~60° at three positions to form a triangle. Functional RNA-protein complexes constructed with this approach could have applications in nanomedicine and synthetic biology.

  5. Solid-State NMR Spectroscopy of Protein Complexes

    PubMed Central

    Sun, Shangjin; Han, Yun; Paramasivam, Sivakumar; Yan, Si; Siglin, Amanda E.; Williams, John C.; Byeon, In-Ja L.; Ahn, Jinwoo; Gronenborn, Angela M.; Polenova, Tatyana

    2016-01-01

    Protein-protein interactions are vital for many biological processes. These interactions often result in the formation of protein assemblies that are large in size, insoluble and difficult to crystallize, and therefore are challenging to study by structure biology techniques, such as single crystal X-ray diffraction and solution NMR spectroscopy. Solid-state NMR (SSNMR) spectroscopy is emerging as a promising technique for studies of such protein assemblies because it is not limited by molecular size, solubility or lack of long-range order. In the past several years, we have applied magic angle spinning SSNMR based methods to study several protein complexes. In this chapter, we discuss the general solid-state NMR methodologies employed for structural and dynamics analyses of protein complexes with specific examples from our work on thioredoxin reassemblies, HIV-1 capsid protein assemblies and microtubule-associated protein assemblies. We present protocols for sample preparation and characterization, pulse sequences, SSNMR spectra collection and data analysis. PMID:22167681

  6. Structural and evolutionary versatility in protein complexes with uneven stoichiometry.

    PubMed

    Marsh, Joseph A; Rees, Holly A; Ahnert, Sebastian E; Teichmann, Sarah A

    2015-03-16

    Proteins assemble into complexes with diverse quaternary structures. Although most heteromeric complexes of known structure have even stoichiometry, a significant minority have uneven stoichiometry--that is, differing numbers of each subunit type. To adopt this uneven stoichiometry, sequence-identical subunits must be asymmetric with respect to each other, forming different interactions within the complex. Here we first investigate the occurrence of uneven stoichiometry, demonstrating that it is common in vitro and is likely to be common in vivo. Next, we elucidate the structural determinants of uneven stoichiometry, identifying six different mechanisms by which it can be achieved. Finally, we study the frequency of uneven stoichiometry across evolution, observing a significant enrichment in bacteria compared with eukaryotes. We show that this arises due to a general increased tendency for bacterial proteins to self-assemble and form homomeric interactions, even within the context of a heteromeric complex.

  7. Conformal Nanopatterning of Extracellular Matrix Proteins onto Topographically Complex Surfaces

    PubMed Central

    Sun, Yan; Jallerat, Quentin; Szymanski, John M.

    2015-01-01

    We report a method for conformal nanopatterning of extracellular matrix proteins onto engineered surfaces independent of underlying microtopography. This enables fibronectin, laminin, and other proteins to be applied to biomaterial surfaces in complex geometries inaccessible using traditional soft lithography techniques. Engineering combinatorial surfaces that integrate topographical and biochemical micropatterns enhances control of the biotic-abiotic interface, used here to understand cardiomyocyte response to competing physical and chemical cues in the microenvironment. PMID:25506720

  8. Biclustering Protein Complex Interactions with a Biclique FindingAlgorithm

    SciTech Connect

    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.

  9. Architecture and function of IFT complex proteins in ciliogenesis.

    PubMed

    Taschner, Michael; Bhogaraju, Sagar; Lorentzen, Esben

    2012-02-01

    Cilia and flagella (interchangeable terms) are evolutionarily conserved organelles found on many different types of eukaryotic cells where they fulfill important functions in motility, sensory reception and signaling. The process of Intraflagellar Transport (IFT) is of central importance for both the assembly and maintenance of cilia, as it delivers building blocks from their site of synthesis in the cell body to the ciliary assembly site at the tip of the cilium. A key player in this process is the multi-subunit IFT-complex, which acts as an adapter between the motor proteins required for movement and the ciliary cargo proteins. Since the discovery of IFT more than 15 years ago, considerable effort has gone into the purification and characterization of the IFT complex proteins. Even though this has led to very interesting findings and has greatly improved our knowledge of the IFT process, we still know very little about the overall architecture of the IFT complex and the specific functions of the various subunits. In this review we will give an update on the knowledge of the structure and function of individual IFT proteins, and the way these proteins interact to form the complex that facilitates IFT. PMID:22118932

  10. Fractal analysis of the structural complexity of the connective tissue in human carotid bodies

    PubMed Central

    Guidolin, Diego; Porzionato, Andrea; Tortorella, Cinzia; Macchi, Veronica; De Caro, Raffaele

    2014-01-01

    The carotid body (CB) may undergo different structural changes during perinatal development, aging, or in response to environmental stimuli. In the previous literature, morphometric approaches to evaluate these changes have considered quantitative first order parameters, such as volumes or densities, while changes in spatial disposition and/or complexity of structural components have not yet been considered. In the present study, different strategies for addressing morphological complexity of CB, apart from the overall amount of each tissue component, were evaluated and compared. In particular, we considered the spatial distribution of connective tissue in the carotid bodies of young control subjects, young opiate-related deaths and aged subjects, through analysis of dispersion (Morisita's index), gray level co-occurrence matrix (entropy, angular second moment, variance, correlation), and fractal analysis (fractal dimension, lacunarity). Opiate-related deaths and aged subjects showed a comparable increase in connective tissue with respect to young controls. However, the Morisita's index (p < 0.05), angular second moment (p < 0.05), fractal dimension (p < 0.01), and lacunarity (p < 0.01) permitted to identify significant differences in the disposition of the connective tissue between these two series. A receiver operating characteristic (ROC) curve was also calculated to evaluate the efficiency of each parameter. The fractal dimension and lacunarity, with areas under the ROC curve of 0.9651 (excellent accuracy) and 0.8835 (good accuracy), respectively, showed the highest discriminatory power. They evidenced higher level of structural complexity in the carotid bodies of opiate-related deaths than old controls, due to more complex branching of intralobular connective tissue. Further analyses will have to consider the suitability of these approaches to address other morphological features of the CB, such as different cell populations, vascularization, and innervation

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

    PubMed

    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

  12. Discovery of host-viral protein complexes during infection

    PubMed Central

    Rowles, Daniell L.; Terhune, Scott S.; Cristea, Ileana M.

    2014-01-01

    Summary Viruses have co-evolved with their hosts, developing effective approaches for hijacking and manipulating host cellular processes. Therefore, for their efficient replication and spread, viruses depend on dynamic and temporally-regulated interactions with host proteins. The rapid identification of host proteins targeted by viral proteins during infection provides significant insights into mechanisms of viral protein function. The resulting discoveries often lead to unique and innovative hypotheses on viral protein function. Here, we describe a robust method for identifying virus-host protein interactions and protein complexes, which we have successfully utilized to characterize spatial-temporal protein interactions during infections with either DNA or RNA viruses, including human cytomegalovirus (HCMV), herpes simplex virus type 1 (HSV-1), pseudorabies virus (PRV), human immunodeficiency virus (HIV-1), Sindbis, and West Nile virus (WNV). This approach involves cryogenic cell lysis, rapid immunoaffinity purification targeting a virus or host protein, followed by identification of associated proteins using mass spectrometry. Like most proteomic approaches, this methodology has evolved over the past few years and continues to evolve. We are presenting here the updated approaches for each step, and discuss alternative strategies allowing for the protocol to be optimized for different biological systems. PMID:23996249

  13. Arabidopsis G-protein interactome reveals connections to cell wall carbohydrates and morphogenesis.

    PubMed

    Klopffleisch, Karsten; Phan, Nguyen; Augustin, Kelsey; Bayne, Robert S; Booker, Katherine S; Botella, Jose R; Carpita, Nicholas C; Carr, Tyrell; Chen, Jin-Gui; Cooke, Thomas Ryan; Frick-Cheng, Arwen; Friedman, Erin J; Fulk, Brandon; Hahn, Michael G; Jiang, Kun; Jorda, Lucia; Kruppe, Lydia; Liu, Chenggang; Lorek, Justine; McCann, Maureen C; Molina, Antonio; Moriyama, Etsuko N; Mukhtar, M Shahid; Mudgil, Yashwanti; Pattathil, Sivakumar; Schwarz, John; Seta, Steven; Tan, Matthew; Temp, Ulrike; Trusov, Yuri; Urano, Daisuke; Welter, Bastian; Yang, Jing; Panstruga, Ralph; Uhrig, Joachim F; Jones, Alan M

    2011-09-27

    The heterotrimeric G-protein complex is minimally composed of Gα, Gβ, and Gγ subunits. In the classic scenario, the G-protein complex is the nexus in signaling from the plasma membrane, where the heterotrimeric G-protein associates with heptahelical G-protein-coupled receptors (GPCRs), to cytoplasmic target proteins called effectors. Although a number of effectors are known in metazoans and fungi, none of these are predicted to exist in their canonical forms in plants. To identify ab initio plant G-protein effectors and scaffold proteins, we screened a set of proteins from the G-protein complex using two-hybrid complementation in yeast. After deep and exhaustive interrogation, we detected 544 interactions between 434 proteins, of which 68 highly interconnected proteins form the core G-protein interactome. Within this core, over half of the interactions comprising two-thirds of the nodes were retested and validated as genuine in planta. Co-expression analysis in combination with phenotyping of loss-of-function mutations in a set of core interactome genes revealed a novel role for G-proteins in regulating cell wall modification.

  14. Arabidopsis G-protein interactome reveals connections to cell wall carbohydrates and morphogenesis

    PubMed Central

    Klopffleisch, Karsten; Phan, Nguyen; Augustin, Kelsey; Bayne, Robert S; Booker, Katherine S; Botella, Jose R; Carpita, Nicholas C; Carr, Tyrell; Chen, Jin-Gui; Cooke, Thomas Ryan; Frick-Cheng, Arwen; Friedman, Erin J; Fulk, Brandon; Hahn, Michael G; Jiang, Kun; Jorda, Lucia; Kruppe, Lydia; Liu, Chenggang; Lorek, Justine; McCann, Maureen C; Molina, Antonio; Moriyama, Etsuko N; Mukhtar, M Shahid; Mudgil, Yashwanti; Pattathil, Sivakumar; Schwarz, John; Seta, Steven; Tan, Matthew; Temp, Ulrike; Trusov, Yuri; Urano, Daisuke; Welter, Bastian; Yang, Jing; Panstruga, Ralph; Uhrig, Joachim F; Jones, Alan M

    2011-01-01

    The heterotrimeric G-protein complex is minimally composed of Gα, Gβ, and Gγ subunits. In the classic scenario, the G-protein complex is the nexus in signaling from the plasma membrane, where the heterotrimeric G-protein associates with heptahelical G-protein-coupled receptors (GPCRs), to cytoplasmic target proteins called effectors. Although a number of effectors are known in metazoans and fungi, none of these are predicted to exist in their canonical forms in plants. To identify ab initio plant G-protein effectors and scaffold proteins, we screened a set of proteins from the G-protein complex using two-hybrid complementation in yeast. After deep and exhaustive interrogation, we detected 544 interactions between 434 proteins, of which 68 highly interconnected proteins form the core G-protein interactome. Within this core, over half of the interactions comprising two-thirds of the nodes were retested and validated as genuine in planta. Co-expression analysis in combination with phenotyping of loss-of-function mutations in a set of core interactome genes revealed a novel role for G-proteins in regulating cell wall modification. PMID:21952135

  15. Arabidopsis G-protein interactome reveals connections to cell wall carbohydrates and morphogenesis

    SciTech Connect

    Klopffleisch, Karsten; Phan, Nguyen; Chen, Jay; Panstruga, Ralph; Uhrig, Joachim; Jones, Alan M

    2011-01-01

    The heterotrimeric G-protein complex is minimally composed of G{alpha}, G{beta}, and G{gamma} subunits. In the classic scenario, the G-protein complex is the nexus in signaling from the plasma membrane, where the heterotrimeric G-protein associates with heptahelical G-protein-coupled receptors (GPCRs), to cytoplasmic target proteins called effectors. Although a number of effectors are known in metazoans and fungi, none of these are predicted to exist in their canonical forms in plants. To identify ab initio plant G-protein effectors and scaffold proteins, we screened a set of proteins from the G-protein complex using two-hybrid complementation in yeast. After deep and exhaustive interrogation, we detected 544 interactions between 434 proteins, of which 68 highly interconnected proteins form the core G-protein interactome. Within this core, over half of the interactions comprising two-thirds of the nodes were retested and validated as genuine in planta. Co-expression analysis in combination with phenotyping of loss-of-function mutations in a set of core interactome genes revealed a novel role for G-proteins in regulating cell wall modification.

  16. Connecting thermal and mechanical protein (un)folding landscapes

    NASA Astrophysics Data System (ADS)

    Sun, Li; Noel, Jeffrey; Sulkowska, Joanna; Levine, Herbert; Onuchic, José

    2015-03-01

    Molecular dynamics simulations supplement single-molecule pulling experiments by providing the possibility of examining the full free energy landscape using many coordinates. Here, we use an all-atom structure-based model to study the force and temperature dependence of the unfolding of the protein filamin by applying force at both termini. The unfolding time-force relation τ(F) indicates that the unfolding behavior can be characterized into three regimes: barrier-limited low- and intermediate-force regimes, and a barrierless high-force regime. Slope changes of τ(F) separate the three regimes. We show that the behavior of τ(F) can be understood from a two-dimensional free energy landscape projected onto the extension X and the fraction of native contacts Q. In the low-force regime, the unfolding rate is roughly force-independent due to the small (even negative) separation in X between the native ensemble and transition state ensemble (TSE). In the intermediate-force regime, force sufficiently separates the TSE from the native ensemble such that τ(F) roughly follows an exponential relation. The TSE becomes increasingly structured with force. The high-force regime is characterized by barrierless unfolding, approaching a time limit of around 10 μs.

  17. Alternate dissociation pathways identified in charge-reduced protein complex ions.

    PubMed

    Pagel, Kevin; Hyung, Suk-Joon; Ruotolo, Brandon T; Robinson, Carol V

    2010-06-15

    Tandem mass spectrometry (MS) of large protein complexes has proven to be capable of assessing the stoichiometry, connectivity, and structural details of multiprotein assemblies. While the utility of tandem MS is without question, a deeper understanding of the mechanism of protein complex dissociation will undoubtedly drive the technology into new areas of enhanced utility and information content. We present here the systematic analysis of the charge state dependent decay of the noncovalently associated complex of human transthyretin, generated by collision-induced dissociation (CID). A crown ether based charge reduction approach was applied to generate intact transthyretin tetramers with charge states ranging from 15+ to 7+. These nine charge states were subsequently analyzed by means of tandem MS and ion mobility spectrometry. Three different charge-dependent mechanistic regimes were identified: (1) common asymmetric dissociation involving ejection of unfolded monomers, (2) expulsion of folded monomers from the intact tetramer, and (3) release of C-terminal peptide fragments from the intact complex. Taken together, the results presented highlight the potential of charge state modulation as a method for directing the course of gas-phase dissociation and unfolding of protein complexes.

  18. A constraint logic programming approach to associate 1D and 3D structural components for large protein complexes.

    PubMed

    Dal Palù, Alessandro; Pontelli, Enrico; He, Jing; Lu, Yonggang

    2007-01-01

    The paper describes a novel framework, constructed using Constraint Logic Programming (CLP) and parallelism, to determine the association between parts of the primary sequence of a protein and alpha-helices extracted from 3D low-resolution descriptions of large protein complexes. The association is determined by extracting constraints from the 3D information, regarding length, relative position and connectivity of helices, and solving these constraints with the guidance of a secondary structure prediction algorithm. Parallelism is employed to enhance performance on large proteins. The framework provides a fast, inexpensive alternative to determine the exact tertiary structure of unknown proteins.

  19. Protein corona - from molecular adsorption to physiological complexity.

    PubMed

    Treuel, Lennart; Docter, Dominic; Maskos, Michael; Stauber, Roland H

    2015-01-01

    In biological environments, nanoparticles are enshrouded by a layer of biomolecules, predominantly proteins, mediating its subsequent interactions with cells. Detecting this protein corona, understanding its formation with regards to nanoparticle (NP) and protein properties, and elucidating its biological implications were central aims of bio-related nano-research throughout the past years. Here, we discuss the mechanistic parameters that are involved in the protein corona formation and the consequences of this corona formation for both, the particle, and the protein. We review consequences of corona formation for colloidal stability and discuss the role of functional groups and NP surface functionalities in shaping NP-protein interactions. We also elaborate the recent advances demonstrating the strong involvement of Coulomb-type interactions between NPs and charged patches on the protein surface. Moreover, we discuss novel aspects related to the complexity of the protein corona forming under physiological conditions in full serum. Specifically, we address the relation between particle size and corona composition and the latest findings that help to shed light on temporal evolution of the full serum corona for the first time. Finally, we discuss the most recent advances regarding the molecular-scale mechanistic role of the protein corona in cellular uptake of NPs.

  20. Connecting thermal and mechanical protein (un)folding landscapes.

    PubMed

    Sun, Li; Noel, Jeffrey K; Sulkowska, Joanna I; Levine, Herbert; Onuchic, José N

    2014-12-16

    Molecular dynamics simulations supplement single-molecule pulling experiments by providing the possibility of examining the full free energy landscape using many coordinates. Here, we use an all-atom structure-based model to study the force and temperature dependence of the unfolding of the protein filamin by applying force at both termini. The unfolding time-force relation τ(F) indicates that the force-induced unfolding behavior of filamin can be characterized into three regimes: barrier-limited low- and intermediate-force regimes, and a barrierless high-force regime. Slope changes of τ(F) separate the three regimes. We show that the behavior of τ(F) can be understood from a two-dimensional free energy landscape projected onto the extension X and the fraction of native contacts Q. In the low-force regime, the unfolding rate is roughly force-independent due to the small (even negative) separation in X between the native ensemble and transition state ensemble (TSE). In the intermediate-force regime, force sufficiently separates the TSE from the native ensemble such that τ(F) roughly follows an exponential relation. This regime is typically explored by pulling experiments. While X may fail to resolve the TSE due to overlap with the unfolded ensemble just below the folding temperature, the overlap is minimal at lower temperatures where experiments are likely to be conducted. The TSE becomes increasingly structured with force, whereas the average order of structural events during unfolding remains roughly unchanged. The high-force regime is characterized by barrierless unfolding, and the unfolding time approaches a limit of ∼10 μs for the highest forces we studied. Finally, a combination of X and Q is shown to be a good reaction coordinate for almost the entire force range. PMID:25517160

  1. Corolla Is a Novel Protein That Contributes to the Architecture of the Synaptonemal Complex of Drosophila

    PubMed Central

    Collins, Kimberly A.; Unruh, Jay R.; Slaughter, Brian D.; Yu, Zulin; Lake, Cathleen M.; Nielsen, Rachel J.; Box, Kimberly S.; Miller, Danny E.; Blumenstiel, Justin P.; Perera, Anoja G.; Malanowski, Kathryn E.; Hawley, R. Scott

    2014-01-01

    In most organisms the synaptonemal complex (SC) connects paired homologs along their entire length during much of meiotic prophase. To better understand the structure of the SC, we aim to identify its components and to determine how each of these components contributes to SC function. Here, we report the identification of a novel SC component in Drosophila melanogaster female oocytes, which we have named Corolla. Using structured illumination microscopy, we demonstrate that Corolla is a component of the central region of the SC. Consistent with its localization, we show by yeast two-hybrid analysis that Corolla strongly interacts with Cona, a central element protein, demonstrating the first direct interaction between two inner-synaptonemal complex proteins in Drosophila. These observations help provide a more complete model of SC structure and function in Drosophila females. PMID:24913682

  2. Study of protein complexes via homology modeling, applied to cysteine proteases and their protein inhibitors.

    PubMed

    Tastan Bishop, Ozlem; Kroon, Matthys

    2011-12-01

    This paper develops and evaluates large-scale calculation of 3D structures of protein complexes by homology modeling as a promising new approach for protein docking. The complexes investigated were papain-like cysteine proteases and their protein inhibitors, which play numerous roles in human and parasitic metabolisms. The structural modeling was performed in two parts. For the first part (evaluation set), nine crystal structure complexes were selected, 1325 homology models of known complexes were rebuilt by various templates including hybrids, allowing an analysis of the factors influencing the accuracy of the models. The important considerations for modeling the interface were protease coverage and inhibitor sequence identity. In the second part (study set), the findings of the evaluation set were used to select appropriate templates to model novel cysteine protease-inhibitor complexes from human and malaria parasites Plasmodium falciparum and Plasmodium vivax. The energy scores, considering the evaluation set, indicate that the models are of high accuracy. PMID:21365221

  3. Immunoprecipitation and Characterization of Membrane Protein Complexes from Yeast

    ERIC Educational Resources Information Center

    Parra-Belky, Karlett; McCulloch, Kathryn; Wick, Nicole; Shircliff, Rebecca; Croft, Nicolas; Margalef, Katrina; Brown, Jamie; Crabill, Todd; Jankord, Ryan; Waldo, Eric

    2005-01-01

    In this undergraduate biochemistry laboratory experiment, the vacuolar ATPase protein complex is purified from yeast cell extracts by doing immunoprecipitations under nondenaturing conditions. Immunoprecipitations are performed using monoclonal antibodies to facilitate data interpretation, and subunits are separated on the basis of their molecular…

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

    PubMed

    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.

  5. Role of loops connecting secondary structure elements in the stabilization of proteins isolated from thermophilic organisms

    PubMed Central

    Balasco, Nicole; Esposito, Luciana; Simone, Alfonso De; Vitagliano, Luigi

    2013-01-01

    It has been recently discovered that the connection of secondary structure elements (ββ-unit, βα- and αβ-units) in proteins follows quite stringent principles regarding the chirality and the orientation of the structural units (Koga et al., Nature 2012;491:222–227). By exploiting these rules, a number of protein scaffolds endowed with a remarkable thermal stability have been designed (Koga et al., Nature 2012;491:222–227). By using structural databases of proteins isolated from either mesophilic or thermophilic organisms, we here investigate the influence of supersecondary associations on the thermal stability of natural proteins. Our results suggest that β-hairpins of proteins from thermophilic organisms are very frequently characterized by shortenings of the loops. Interestingly, this shortening leads to states that display a very strong preference for the most common connectivity of the strands observed in native protein hairpins. The abundance of selective states in these proteins suggests that they may achieve a high stability by adopting a strategy aimed to reduce the possible conformations of the unfolded ensemble. In this scenario, our data indicate that the shortening is effective if it increases the adherence to these rules. We also show that this mechanism may operate in the stabilization of well-known protein folds (thioredoxin and RNase A). These findings suggest that future investigations aimed at defining mechanism of protein stabilization should also consider these effects. PMID:23661276

  6. Automated two-dimensional liquid chromatographic system for mapping proteins in highly complex mixtures.

    PubMed

    Isobe, T; Uchida, K; Taoka, M; Shinkai, F; Manabe, T; Okuyama, T

    1991-12-27

    An automated two-dimensional liquid chromatographic system was developed for systematic protein separations which could serve for analytical mapping and preparative separations of proteins. The system applies the principles of the column-switching technique, and consists of two different columns connected in tandem through an electrical column switching valve, two pumping systems to operate each column independently and a system controller to perform sequential chromatography on the two columns. A protein mixture is applied to the first-dimensional anion-exchange column and is separated by stepwise elution with an increasing sodium chloride concentration. The eluent is introduced directly to the second-dimensional reversed-phase column, and is further separated by gradient elution with an increasing acetonitrile concentration. The two elution stages are synchronized by a computer program. By this system, very complex protein mixtures such as crude cerebellar extracts were resolved reproducibly into ca. 200 peaks within 12 h. The method can be used for the total analysis of proteins in various tissues and cells without complicated premanupulation of samples, and allows the simultaneous analysis of a protein isolated by chromatography. The isolated protein is most suitable for use in the strategy of protein and gene sequence analysis.

  7. Protein import and the origin of red complex plastids.

    PubMed

    Gould, Sven B; Maier, Uwe-G; Martin, William F

    2015-06-15

    The number and nature of endosymbioses involving red algal endosymbionts are debated. Gene phylogenies have become the most popular tool to untangle this issue, but they deliver conflicting results. As gene and lineage sampling has increased, so have both the number of conflicting trees and the number of suggestions in the literature for multiple tertiary, and even quaternary, symbioses that might reconcile the tree conflicts. Independent lines of evidence that can address the issue are needed. Here we summarize the mechanism and machinery of protein import into complex red plastids. The process involves protein translocation machinery, known as SELMA, that arose once in evolution, that facilitates protein import across the second outermost of the four plastid membranes, and that is always targeted specifically to that membrane, regardless of where it is encoded today. It is widely accepted that the unity of protein import across the two membranes of primary plastids is strong evidence for their single cyanobacterial origin. Similarly, the unity of SELMA-dependent protein import across the second outermost plastid membrane constitutes strong evidence for the existence of a single red secondary endosymbiotic event at the common origin of all red complex plastids. We furthermore propose that the two outer membranes of red complex plastids are derived from host endoplasmic reticulum in the initial red secondary endosymbiotic event. PMID:26079086

  8. Protein import and the origin of red complex plastids.

    PubMed

    Gould, Sven B; Maier, Uwe-G; Martin, William F

    2015-06-15

    The number and nature of endosymbioses involving red algal endosymbionts are debated. Gene phylogenies have become the most popular tool to untangle this issue, but they deliver conflicting results. As gene and lineage sampling has increased, so have both the number of conflicting trees and the number of suggestions in the literature for multiple tertiary, and even quaternary, symbioses that might reconcile the tree conflicts. Independent lines of evidence that can address the issue are needed. Here we summarize the mechanism and machinery of protein import into complex red plastids. The process involves protein translocation machinery, known as SELMA, that arose once in evolution, that facilitates protein import across the second outermost of the four plastid membranes, and that is always targeted specifically to that membrane, regardless of where it is encoded today. It is widely accepted that the unity of protein import across the two membranes of primary plastids is strong evidence for their single cyanobacterial origin. Similarly, the unity of SELMA-dependent protein import across the second outermost plastid membrane constitutes strong evidence for the existence of a single red secondary endosymbiotic event at the common origin of all red complex plastids. We furthermore propose that the two outer membranes of red complex plastids are derived from host endoplasmic reticulum in the initial red secondary endosymbiotic event.

  9. The multimodal connectivity of the hippocampal complex in auditory and visual hallucinations.

    PubMed

    Amad, A; Cachia, A; Gorwood, P; Pins, D; Delmaire, C; Rolland, B; Mondino, M; Thomas, P; Jardri, R

    2014-02-01

    Hallucinations constitute one of the most representative and disabling symptoms of schizophrenia. Several Magnetic Resonance Imaging (MRI) findings support the hypothesis that distinct patterns of connectivity, particularly within networks involving the hippocampal complex (HC), could be associated with different hallucinatory modalities. The aim of this study was to investigate HC connectivity as a function of the hallucinatory modality, that is, auditory or visual. Two carefully selected subgroups of schizophrenia patients with only auditory hallucinations (AH) or with audio-visual hallucinations (A+VH) were compared using the following three complementary multimodal MRI methods: resting state functional MRI, diffusion MRI and structural MRI were used to analyze seed-based Functional Connectivity (sb-FC), Tract-Based Spatial Statistics (TBSS) and shape analysis, respectively. Sb-FC was significantly higher between the HC, the medial prefrontal cortex (mPFC) and the caudate nuclei in A+VH patients compared with the AH group. Conversely, AH patients exhibited a higher sb-FC between the HC and the thalamus in comparison with the A+VH group. In the A+VH group, TBSS showed specific higher white matter connectivity in the pathways connecting the HC with visual areas, such as the forceps major and the inferior-fronto-occipital fasciculus than in the AH group. Finally, shape analysis showed localized hippocampal hypertrophy in the A+VH group. Functional results support the fronto-limbic dysconnectivity hypothesis of schizophrenia, while specific structural findings indicate that plastic changes are associated with hallucinations. Together, these results suggest that there are distinct connectivity patterns in patients with schizophrenia that depend on the sensory-modality, with specific involvement of the HC in visual hallucinations.

  10. DOCK/PIERR: web server for structure prediction of protein-protein complexes.

    PubMed

    Viswanath, Shruthi; Ravikant, D V S; Elber, Ron

    2014-01-01

    In protein docking we aim to find the structure of the complex formed when two proteins interact. Protein-protein interactions are crucial for cell function. Here we discuss the usage of DOCK/PIERR. In DOCK/PIERR, a uniformly discrete sampling of orientations of one protein with respect to the other, are scored, followed by clustering, refinement, and reranking of structures. The novelty of this method lies in the scoring functions used. These are obtained by examining hundreds of millions of correctly and incorrectly docked structures, using an algorithm based on mathematical programming, with provable convergence properties.

  11. Folding Behaviors of Protein (Lysozyme) Confined in Polyelectrolyte Complex Micelle.

    PubMed

    Wu, Fu-Gen; Jiang, Yao-Wen; Chen, Zhan; Yu, Zhi-Wu

    2016-04-19

    The folding/unfolding behavior of proteins (enzymes) in confined space is important for their properties and functions, but such a behavior remains largely unexplored. In this article, we reported our finding that lysozyme and a double hydrophilic block copolymer, methoxypoly(ethylene glycol)5K-block-poly(l-aspartic acid sodium salt)10 (mPEG(5K)-b-PLD10), can form a polyelectrolyte complex micelle with a particle size of ∼30 nm, as verified by dynamic light scattering and transmission electron microscopy. The unfolding and refolding behaviors of lysozyme molecules in the presence of the copolymer were studied by microcalorimetry and circular dichroism spectroscopy. Upon complex formation with mPEG(5K)-b-PLD10, lysozyme changed from its initial native state to a new partially unfolded state. Compared with its native state, this copolymer-complexed new folding state of lysozyme has different secondary and tertiary structures, a decreased thermostability, and significantly altered unfolding/refolding behaviors. It was found that the native lysozyme exhibited reversible unfolding and refolding upon heating and subsequent cooling, while lysozyme in the new folding state (complexed with the oppositely charged PLD segments of the polymer) could unfold upon heating but could not refold upon subsequent cooling. By employing the heating-cooling-reheating procedure, the prevention of complex formation between lysozyme and polymer due to the salt screening effect was observed, and the resulting uncomplexed lysozyme regained its proper unfolding and refolding abilities upon heating and subsequent cooling. Besides, we also pointed out the important role the length of the PLD segment played during the formation of micelles and the monodispersity of the formed micelles. Furthermore, the lysozyme-mPEG(5K)-b-PLD10 mixtures prepared in this work were all transparent, without the formation of large aggregates or precipitates in solution as frequently observed in other protein

  12. Role of protein fluctuation correlations in electron transfer in photosynthetic complexes.

    PubMed

    Nesterov, Alexander I; Berman, Gennady P

    2015-04-01

    We consider the dependence of the electron transfer in photosynthetic complexes on correlation properties of random fluctuations of the protein environment. The electron subsystem is modeled by a finite network of connected electron (exciton) sites. The fluctuations of the protein environment are modeled by random telegraph processes, which act either collectively (correlated) or independently (uncorrelated) on the electron sites. We derived an exact closed system of first-order linear differential equations with constant coefficients, for the average density matrix elements and for their first moments. Under some conditions, we obtained analytic expressions for the electron transfer rates and found the range of parameters for their applicability by comparing with the exact numerical simulations. We also compared the correlated and uncorrelated regimes and demonstrated numerically that the uncorrelated fluctuations of the protein environment can, under some conditions, either increase or decrease the electron transfer rates. PMID:25974522

  13. Computational large-scale mapping of protein-protein interactions using structural complexes.

    PubMed

    Shoemaker, Benjamin; Wuchty, Stefan; Panchenko, Anna R

    2013-01-01

    Although the identification of protein interactions by high-throughput methods progresses at a fast pace, "interactome" datasets still suffer from high rates of false positives and low coverage. To map the interactome of any organism, this unit presents a computational framework to predict protein-protein or gene-gene interactions utilizing experimentally determined evidence of structural complexes, atomic details of binding interfaces and evolutionary conservation.

  14. Complexity of Low-Frequency BOLD Fluctuations Covaries with Local Connectivity

    PubMed Central

    Anderson, Jeffrey S.; Zielinski, Brandon A.; Nielsen, Jared A.; Ferguson, Michael A.

    2014-01-01

    Very low frequency blood oxygen level dependent (BOLD) fluctuations have emerged as a valuable tool for describing brain anatomy, neuropathology, and development. Such fluctuations exhibit power law frequency dynamics, with largest amplitude at lowest frequencies. The biophysical mechanisms generating such fluctuations are poorly understood. Using publicly available data from 1019 subjects of age 7-30, we show that BOLD fluctuations exhibit temporal complexity that is linearly related to local connectivity (regional homogeneity), consistently and significantly covarying across subjects and across gray matter regions. This relationship persisted independently of covariance with gray matter density or standard deviation of BOLD signal. During late neurodevelopment, BOLD fluctuations were unchanged with age in association cortex while becoming more random throughout the rest of the brain. These data suggest that local interconnectivity may play a key role in establishing the complexity of low frequency BOLD fluctuations underlying functional MRI connectivity. Stable low frequency power dynamics may emerge through segmentation and integration of connectivity during development of distributed large-scale brain networks. PMID:23417795

  15. Abnormal EEG Complexity and Functional Connectivity of Brain in Patients with Acute Thalamic Ischemic Stroke

    PubMed Central

    Liu, Shuang; Guo, Jie; Meng, Jiayuan; Wang, Zhijun; Yao, Yang; Yang, Jiajia; Qi, Hongzhi; Ming, Dong

    2016-01-01

    Ischemic thalamus stroke has become a serious cardiovascular and cerebral disease in recent years. To date the existing researches mostly concentrated on the power spectral density (PSD) in several frequency bands. In this paper, we investigated the nonlinear features of EEG and brain functional connectivity in patients with acute thalamic ischemic stroke and healthy subjects. Electroencephalography (EEG) in resting condition with eyes closed was recorded for 12 stroke patients and 11 healthy subjects as control group. Lempel-Ziv complexity (LZC), Sample Entropy (SampEn), and brain network using partial directed coherence (PDC) were calculated for feature extraction. Results showed that patients had increased mean LZC and SampEn than the controls, which implied the stroke group has higher EEG complexity. For the brain network, the stroke group displayed a trend of weaker cortical connectivity, which suggests a functional impairment of information transmission in cortical connections in stroke patients. These findings suggest that nonlinear analysis and brain network could provide essential information for better understanding the brain dysfunction in the stroke and assisting monitoring or prognostication of stroke evolution. PMID:27403202

  16. Transmembrane protein 147 (TMEM147) is a novel component of the Nicalin-NOMO protein complex.

    PubMed

    Dettmer, Ulf; Kuhn, Peer-Hendrik; Abou-Ajram, Claudia; Lichtenthaler, Stefan F; Krüger, Marcus; Kremmer, Elisabeth; Haass, Christian; Haffner, Christof

    2010-08-20

    Nicastrin and its relative Nicalin (Nicastrin-like protein) are both members of larger protein complexes, namely gamma-secretase and the Nicalin-NOMO (Nodal modulator) complex. The gamma-secretase complex, which contains Presenilin, APH-1, and PEN-2 in addition to Nicastrin, catalyzes the proteolytic cleavage of the transmembrane domain of various proteins including the beta-amyloid precursor protein and Notch. Nicalin and its binding partner NOMO form a complex that was shown to modulate Nodal signaling in developing zebrafish embryos. Because its experimentally determined native size (200-220 kDa) could not be satisfyingly explained by the molecular masses of Nicalin (60 kDa) and NOMO (130 kDa), we searched in affinity-purified complex preparations for additional components in the low molecular mass range. A approximately 22-kDa protein was isolated and identified by mass spectrometry as transmembrane protein 147 (TMEM147), a novel, highly conserved membrane protein with a putative topology similar to APH-1. Like Nicalin and NOMO, it localizes to the endoplasmic reticulum and is expressed during early zebrafish development. Overexpression and knockdown experiments in cultured cells demonstrate a close relationship between the three proteins and suggest that they are components of the same complex. We present evidence that, similar to gamma-secretase, its assembly is hierarchical starting with the formation of a Nicalin-NOMO intermediate. Nicalin appears to represent the limiting factor regulating the assembly rate by stabilizing the other two components. We conclude that TMEM147 is a novel core component of the Nicalin-NOMO complex, further emphasizing its similarity with gamma-secretase. PMID:20538592

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

    PubMed

    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

  18. Modeling of Protein Binary Complexes Using Structural Mass Spectrometry Data

    SciTech Connect

    Amisha Kamal,J.; Chance, M.

    2008-01-01

    In this article, we describe a general approach to modeling the structure of binary protein complexes using structural mass spectrometry data combined with molecular docking. In the first step, hydroxyl radical mediated oxidative protein footprinting is used to identify residues that experience conformational reorganization due to binding or participate in the binding interface. In the second step, a three-dimensional atomic structure of the complex is derived by computational modeling. Homology modeling approaches are used to define the structures of the individual proteins if footprinting detects significant conformational reorganization as a function of complex formation. A three-dimensional model of the complex is constructed from these binary partners using the ClusPro program, which is composed of docking, energy filtering, and clustering steps. Footprinting data are used to incorporate constraints--positive and/or negative--in the docking step and are also used to decide the type of energy filter--electrostatics or desolvation--in the successive energy-filtering step. By using this approach, we examine the structure of a number of binary complexes of monomeric actin and compare the results to crystallographic data. Based on docking alone, a number of competing models with widely varying structures are observed, one of which is likely to agree with crystallographic data. When the docking steps are guided by footprinting data, accurate models emerge as top scoring. We demonstrate this method with the actin/gelsolin segment-1 complex. We also provide a structural model for the actin/cofilin complex using this approach which does not have a crystal or NMR structure.

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

    PubMed

    Chakravarty, Devlina; Janin, Joël; Robert, Charles H; Chakrabarti, Pinak

    2015-11-01

    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.

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

    PubMed Central

    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

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

    PubMed

    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.

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

    PubMed

    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. PMID:26806547

  3. Mechanism of action of regulatory proteins encoded by complex retroviruses.

    PubMed Central

    Cullen, B R

    1992-01-01

    Complex retroviruses are distinguished by their ability to control the expression of their gene products through the action of virally encoded regulatory proteins. These viral gene products modulate both the quantity and the quality of viral gene expression through regulation at both the transcriptional and posttranscriptional levels. The most intensely studied retroviral regulatory proteins, termed Tat and Rev, are encoded by the prototypic complex retrovirus human immunodeficiency virus type 1. However, considerable information also exists on regulatory proteins encoded by human T-cell leukemia virus type I, as well as several other human and animal complex retroviruses. In general, these data demonstrate that retrovirally encoded transcriptional trans-activators can exert a similar effect by several very different mechanisms. In contrast, posttranscriptional regulation of retroviral gene expression appears to occur via a single pathway that is probably dependent on the recruitment of a highly conserved cellular cofactor. These two shared regulatory pathways are proposed to be critical to the ability of complex retroviruses to establish chronic infections in the face of an ongoing host immune response. Images PMID:1406488

  4. An update on the connections of the ventral mesencephalic dopaminergic complex.

    PubMed

    Yetnikoff, L; Lavezzi, H N; Reichard, R A; Zahm, D S

    2014-12-12

    This review covers the intrinsic organization and afferent and efferent connections of the midbrain dopaminergic complex, comprising the substantia nigra, ventral tegmental area and retrorubral field, which house, respectively, the A9, A10 and A8 groups of nigrostriatal, mesolimbic and mesocortical dopaminergic neurons. In addition, A10dc (dorsal, caudal) and A10rv (rostroventral) extensions into, respectively, the ventrolateral periaqueductal gray and supramammillary nucleus are discussed. Associated intrinsic and extrinsic connections of the midbrain dopaminergic complex that utilize gamma-aminobutyric acid (GABA), glutamate and neuropeptides and various co-expressed combinations of these compounds are considered in conjunction with the dopamine-containing systems. A framework is provided for understanding the organization of massive afferent systems descending and ascending to the midbrain dopaminergic complex from the telencephalon and brainstem, respectively. Within the context of this framework, the basal ganglia direct and indirect output pathways are treated in some detail. Findings from rodent brain are briefly compared with those from primates, including humans. Recent literature is emphasized, including traditional experimental neuroanatomical and modern gene transfer and optogenetic studies. An attempt was made to provide sufficient background and cite a representative sampling of earlier primary papers and reviews so that people new to the field may find this to be a relatively comprehensive treatment of the subject. PMID:24735820

  5. A Bacillus megaterium System for the Production of Recombinant Proteins and Protein Complexes.

    PubMed

    Biedendieck, Rebekka

    2016-01-01

    For many years the Gram-positive bacterium Bacillus megaterium has been used for the production and secretion of recombinant proteins. For this purpose it was systematically optimized. Plasmids with different inducible promoter systems, with different compatible origins, with small tags for protein purification and with various specific signals for protein secretion were combined with genetically improved host strains. Finally, the development of appropriate cultivation conditions for the production strains established this organism as a bacterial cell factory even for large proteins. Along with the overproduction of individual proteins the organism is now also used for the simultaneous coproduction of up to 14 recombinant proteins, multiple subsequently interacting or forming protein complexes. Some of these recombinant strains are successfully used for bioconversion or the biosynthesis of valuable components including vitamins. The titers in the g per liter scale for the intra- and extracellular recombinant protein production prove the high potential of B. megaterium for industrial applications. It is currently further enhanced for the production of recombinant proteins and multi-subunit protein complexes using directed genetic engineering approaches based on transcriptome, proteome, metabolome and fluxome data. PMID:27165321

  6. A Bacillus megaterium System for the Production of Recombinant Proteins and Protein Complexes.

    PubMed

    Biedendieck, Rebekka

    2016-01-01

    For many years the Gram-positive bacterium Bacillus megaterium has been used for the production and secretion of recombinant proteins. For this purpose it was systematically optimized. Plasmids with different inducible promoter systems, with different compatible origins, with small tags for protein purification and with various specific signals for protein secretion were combined with genetically improved host strains. Finally, the development of appropriate cultivation conditions for the production strains established this organism as a bacterial cell factory even for large proteins. Along with the overproduction of individual proteins the organism is now also used for the simultaneous coproduction of up to 14 recombinant proteins, multiple subsequently interacting or forming protein complexes. Some of these recombinant strains are successfully used for bioconversion or the biosynthesis of valuable components including vitamins. The titers in the g per liter scale for the intra- and extracellular recombinant protein production prove the high potential of B. megaterium for industrial applications. It is currently further enhanced for the production of recombinant proteins and multi-subunit protein complexes using directed genetic engineering approaches based on transcriptome, proteome, metabolome and fluxome data.

  7. Efficient Prediction of Co-Complexed Proteins Based on Coevolution

    PubMed Central

    de Vienne, Damien M.; Azé, Jérôme

    2012-01-01

    The prediction of the network of protein-protein interactions (PPI) of an organism is crucial for the understanding of biological processes and for the development of new drugs. Machine learning methods have been successfully applied to the prediction of PPI in yeast by the integration of multiple direct and indirect biological data sources. However, experimental data are not available for most organisms. We propose here an ensemble machine learning approach for the prediction of PPI that depends solely on features independent from experimental data. We developed new estimators of the coevolution between proteins and combined them in an ensemble learning procedure. We applied this method to a dataset of known co-complexed proteins in Escherichia coli and compared it to previously published methods. We show that our method allows prediction of PPI with an unprecedented precision of 95.5% for the first 200 sorted pairs of proteins compared to 28.5% on the same dataset with the previous best method. A close inspection of the best predicted pairs allowed us to detect new or recently discovered interactions between chemotactic components, the flagellar apparatus and RNA polymerase complexes in E. coli. PMID:23152796

  8. The two faces of Alba: the evolutionary connection between proteins participating in chromatin structure and RNA metabolism

    PubMed Central

    Aravind, L; Iyer, Lakshminarayan M; Anantharaman, Vivek

    2003-01-01

    Background There is considerable heterogeneity in the phyletic patterns of major chromosomal DNA-binding proteins in archaea. Alba is a well-characterized chromosomal protein from the crenarchaeal genus Sulfolobus. While Alba has been detected in most archaea and some eukaryotic taxa, its exact functions in these taxa are not clear. Here we use comparative genomics and sequence profile analysis to predict potential alternative functions of the Alba proteins. Results Using sequence-profile searches, we were able to unify the Alba proteins with RNase P/MRP subunit Rpp20/Pop7, human RNase P subunit Rpp25, and the ciliate Mdp2 protein, which is implicated in macronuclear development. The Alba superfamily contains two eukaryote-specific families and one archaeal family. We present different lines of evidence to show that both eukaryotic families perform functions related to RNA metabolism. Several members of one of the eukaryotic families, typified by Mdp2, are combined in the same polypeptide with RNA-binding RGG repeats. We also investigated the relationships of the unified Alba superfamily within the ancient RNA-binding IF3-C fold, and show that it is most closely related to other RNA-binding members of this fold, such as the YhbY and IF3-C superfamilies. Based on phyletic patterns and the principle of phylogenetic bracketing, we predict that at least some of the archaeal members may also possess a role in RNA metabolism. Conclusions The Alba superfamily proteins appear to have originated as RNA-binding proteins which formed various ribonucleoprotein complexes, probably including RNase P. It was recruited as a chromosomal protein possibly only within the crenarchaeal lineage. The evolutionary connections reported here suggest how a diversity of functions based on a common biochemical basis emerged in proteins of the Alba superfamily. PMID:14519199

  9. Detecting protein complexes from active protein interaction networks constructed with dynamic gene expression profiles

    PubMed Central

    2013-01-01

    Background Protein interaction networks (PINs) are known to be useful to detect protein complexes. However, most available PINs are static, which cannot reflect the dynamic changes in real networks. At present, some researchers have tried to construct dynamic networks by incorporating time-course (dynamic) gene expression data with PINs. However, the inevitable background noise exists in the gene expression array, which could degrade the quality of dynamic networkds. Therefore, it is needed to filter out contaminated gene expression data before further data integration and analysis. Results Firstly, we adopt a dynamic model-based method to filter noisy data from dynamic expression profiles. Then a new method is proposed for identifying active proteins from dynamic gene expression profiles. An active protein at a time point is defined as the protein the expression level of whose corresponding gene at that time point is higher than a threshold determined by a standard variance involved threshold function. Furthermore, a noise-filtered active protein interaction network (NF-APIN) is constructed. To demonstrate the efficiency of our method, we detect protein complexes from the NF-APIN, compared with those from other dynamic PINs. Conclusion A dynamic model based method can effectively filter out noises in dynamic gene expression data. Our method to compute a threshold for determining the active time points of noise-filtered genes can make the dynamic construction more accuracy and provide a high quality framework for network analysis, such as protein complex prediction. PMID:24565281

  10. The mammalian autophagy initiator complex contains 2 HORMA domain proteins

    PubMed Central

    Michel, Max; Schwarten, Melanie; Decker, Christina; Nagel-Steger, Luitgard; Willbold, Dieter; Weiergräber, Oliver H

    2015-01-01

    ATG101 is an essential component of the ULK complex responsible for initiating cellular autophagy in mammalian cells; its 3-dimensional structure and molecular function, however, are currently unclear. Here we present the X-ray structure of human ATG101. The protein displays an open HORMA domain fold. Both structural properties and biophysical evidence indicate that ATG101 is locked in this conformation, in contrast to the prototypical HORMA domain protein MAD2. Moreover, we discuss a potential mode of dimerization with ATG13 as a fundamental aspect of ATG101 function. PMID:26236954

  11. Dark dyes-bright complexes: fluorogenic protein labeling.

    PubMed

    Bruchez, Marcel P

    2015-08-01

    Complexes formed between organic dyes and genetically encoded proteins combine the advantages of stable and tunable fluorescent molecules and targetable, biologically integrated labels. To overcome the challenges imposed by labeling with bright fluorescent dyes, a number of approaches now exploit chemical or environmental changes to control the properties of a bound dye, converting dyes from a weakly fluorescent state to a bright, easily detectable complex. Optimized, such approaches avoid the need for removal of unbound dyes, facilitate rapid and simple assays in cultured cells and enable hybrid labeling to function more robustly in living model organisms.

  12. Protein fragment bimolecular fluorescence complementation analyses for the in vivo study of protein-protein interactions and cellular protein complex localizations

    PubMed Central

    Waadt, Rainer; Schlücking, Kathrin; Schroeder, Julian I.; Kudla, Jörg

    2014-01-01

    Summary The analyses of protein-protein interactions is crucial for understanding cellular processes including signal transduction, protein trafficking and movement. Protein fragment complementation assays are based on the reconstitution of protein function when non-active protein fragments are brought together by interacting proteins that were genetically fused to these protein fragments. Bimolecular fluorescence complementation (BiFC) relies on the reconstitution of fluorescent proteins and enables both the analysis of protein-protein interactions and the visualization of protein complex formations in vivo. Transient expression of proteins is a convenient approach to study protein functions in planta or in other organisms, and minimizes the need for time-consuming generation of stably expressing transgenic organisms. Here we describe protocols for BiFC analyses in Nicotiana benthamiana and Arabidopsis thaliana leaves transiently transformed by Agrobacterium infiltration. Further we discuss different BiFC applications and provide examples for proper BiFC analyses in planta. PMID:24057390

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

    PubMed

    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.

  14. Supercharging Protein Complexes from Aqueous Solution Disrupts their Native Conformations

    NASA Astrophysics Data System (ADS)

    Sterling, Harry J.; Kintzer, Alexander F.; Feld, Geoffrey K.; Cassou, Catherine A.; Krantz, Bryan A.; Williams, Evan R.

    2012-02-01

    The effects of aqueous solution supercharging on the solution- and gas-phase structures of two protein complexes were investigated using traveling-wave ion mobility-mass spectrometry (TWIMS-MS). Low initial concentrations of m-nitrobenzyl alcohol ( m-NBA) in the electrospray ionization (ESI) solution can effectively increase the charge of concanavalin A dimers and tetramers, but at higher m-NBA concentrations, the increases in charge are accompanied by solution-phase dissociation of the dimers and up to a ~22% increase in the collision cross section (CCS) of the tetramers. With just 0.8% m-NBA added to the ESI solution of a ~630 kDa anthrax toxin octamer complex, the average charge is increased by only ~4% compared with the "native" complex, but it is sufficiently destabilized so that extensive gas-phase fragmentation occurs in the relatively high pressure regions of the TWIMS device. Anthrax toxin complexes exist in either a prechannel or a transmembrane channel state. With m-NBA, the prechannel state of the complex has the same CCS/charge ratio in the gas phase as the transmembrane channel state of the same complex formed without m-NBA, yet undergoes extensive dissociation, indicating that destabilization from supercharging occurs in the ESI droplet prior to ion formation and is not a result of Coulombic destabilization in the gas phase as a result of higher charging. These results demonstrate that the supercharging of large protein complexes is the result of conformational changes induced by the reagents in the ESI droplets, where enrichment of the supercharging reagent during droplet evaporation occurs.

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

    PubMed

    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.

  16. Rational stabilization of complex proteins: a divide and combine approach

    PubMed Central

    Lamazares, Emilio; Clemente, Isabel; Bueno, Marta; Velázquez-Campoy, Adrián; Sancho, Javier

    2015-01-01

    Increasing the thermostability of proteins is often crucial for their successful use as analytic, synthetic or therapeutic tools. Most rational thermostabilization strategies were developed on small two-state proteins and, unsurprisingly, they tend to fail when applied to the much more abundant, larger, non-fully cooperative proteins. We show that the key to stabilize the latter is to know the regions of lower stability. To prove it, we have engineered apoflavodoxin, a non-fully cooperative protein on which previous thermostabilizing attempts had failed. We use a step-wise combination of structure-based, rationally-designed, stabilizing mutations confined to the less stable structural region, and obtain variants that, according to their van't Hoff to calorimetric enthalpy ratios, exhibit fully-cooperative thermal unfolding with a melting temperature of 75°C, 32 degrees above the lower melting temperature of the non-cooperative wild type protein. The ideas introduced here may also be useful for the thermostabilization of complex proteins through formulation or using specific stabilizing ligands (e.g. pharmacological chaperones). PMID:25774740

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

    PubMed

    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.

  18. Connectivity, small islands and large distances: the Cellana strigilis limpet complex in the Southern Ocean.

    PubMed

    Reisser, Céline M O; Wood, Ann R; Bell, James J; Gardner, Jonathan P A

    2011-08-01

    The Southern Ocean contains some of the most isolated islands on Earth, and fundamental questions remain regarding their colonization and the connectivity of their coastal biotas. Here, we conduct a genetic investigation into the Cellana strigilis (limpet) complex that was originally classified based on morphological characters into six subspecies, five of which are endemic to the New Zealand (NZ) subantarctic and Chatham islands (44-52°S). Previous genetic analyses of C. strigilis from six of the seven island groups revealed two lineages with little or no within-lineage variation. We analysed C. strigilis samples from all seven island groups using two mitochondrial (COI and 16S), one nuclear (ATPase β) and 58 loci from four randomly amplified polymorphic DNA markers (RAPDs) and confirmed the existence of two distinct lineages. The pronounced genetic structuring within each lineage and the presence of private haplotypes in individual islands are the result of little genetic connectivity and therefore very high self-recruitment. This study supports the significance of the subantarctic islands as refugia during the last glacial maximum and adds to the knowledge of contemporary population connectivity among coastal populations of remote islands in large oceans and the distance barrier to gene flow that exists in the sea (despite its continuous medium) for most taxa.

  19. DMS Footprinting of Structured RNAs and RNA-Protein Complexes

    PubMed Central

    Tijerina, Pilar; Mohr, Sabine; Russell, Rick

    2008-01-01

    We describe a protocol in which dimethyl sulfate (DMS) modification of the base-pairing faces of unpaired adenosine and cytidine nucleotides is used for structural analysis of RNAs and RNA-protein complexes (RNPs). The protocol is optimized for RNAs of small to moderate size (≤500 nucleotides). The RNA or RNP is first exposed to DMS under conditions that promote formation of the folded structure or complex, as well as ‘control’ conditions that do not allow folding or complex formation. The positions and extents of modification are then determined by primer extension, polyacrylamide gel electrophoresis (PAGE), and quantitative analysis. From changes in the extent of modification upon folding or protein binding (appearance of a ‘footprint’), it is possible to detect local changes in RNA secondary and tertiary structure, as well as the formation of RNA-protein contacts. This protocol takes 1.5–3 days to complete, depending on the type of analysis used. PMID:17948004

  20. Connected Component Labeling algorithm for very complex and high-resolution images on an FPGA platform

    NASA Astrophysics Data System (ADS)

    Schwenk, Kurt; Huber, Felix

    2015-10-01

    Connected Component Labeling (CCL) is a basic algorithm in image processing and an essential step in nearly every application dealing with object detection. It groups together pixels belonging to the same connected component (e.g. object). Special architectures such as ASICs, FPGAs and GPUs were utilised for achieving high data throughput, primarily for video processing. In this article, the FPGA implementation of a CCL method is presented, which was specially designed to process high resolution images with complex structure at high speed, generating a label mask. In general, CCL is a dynamic task and therefore not well suited for parallelisation, which is needed to achieve high processing speed with an FPGA. Facing this issue, most of the FPGA CCL implementations are restricted to low or medium resolution images (≤ 2048 ∗ 2048 pixels) with lower complexity, where the fastest implementations do not create a label mask. Instead, they extract object features like size and position directly, which can be realized with high performance and perfectly suits the need for many video applications. Since these restrictions are incompatible with the requirements to label high resolution images with highly complex structures and the need for generating a label mask, a new approach was required. The CCL method presented in this work is based on a two-pass CCL algorithm, which was modified with respect to low memory consumption and suitability for an FPGA implementation. Nevertheless, since not all parts of CCL can be parallelised, a stop-and-go high-performance pipeline processing CCL module was designed. The algorithm, the performance and the hardware requirements of a prototype implementation are presented. Furthermore, a clock-accurate runtime analysis is shown, which illustrates the dependency between processing speed and image complexity in detail. Finally, the performance of the FPGA implementation is compared with that of a software implementation on modern embedded

  1. A secretory kinase complex regulates extracellular protein phosphorylation

    PubMed Central

    Cui, Jixin; Xiao, Junyu; Tagliabracci, Vincent S; Wen, Jianzhong; Rahdar, Meghdad; Dixon, Jack E

    2015-01-01

    Although numerous extracellular phosphoproteins have been identified, the protein kinases within the secretory pathway have only recently been discovered, and their regulation is virtually unexplored. Fam20C is the physiological Golgi casein kinase, which phosphorylates many secreted proteins and is critical for proper biomineralization. Fam20A, a Fam20C paralog, is essential for enamel formation, but the biochemical function of Fam20A is unknown. Here we show that Fam20A potentiates Fam20C kinase activity and promotes the phosphorylation of enamel matrix proteins in vitro and in cells. Mechanistically, Fam20A is a pseudokinase that forms a functional complex with Fam20C, and this complex enhances extracellular protein phosphorylation within the secretory pathway. Our findings shed light on the molecular mechanism by which Fam20C and Fam20A collaborate to control enamel formation, and provide the first insight into the regulation of secretory pathway phosphorylation. DOI: http://dx.doi.org/10.7554/eLife.06120.001 PMID:25789606

  2. Interactions between the kinetochore complex and the protein kinase A pathway in Saccharomyces cerevisiae.

    PubMed

    Ma, Lina; Ho, Krystina; Piggott, Nina; Luo, Zongli; Measday, Vivien

    2012-07-01

    The kinetochore is a large structure composed of multiple protein subcomplexes that connect chromosomes to spindle microtubules to enable accurate chromosome segregation. Significant advances have been made in the identification of kinetochore proteins and elucidation of kinetochore structure; however, comparatively little is known about how cellular signals integrate with kinetochore function. In the budding yeast Saccharomyces cerevisiae, the cyclic AMP protein kinase A signaling pathway promotes cellular growth in response to glucose. In this study, we find that decreasing protein kinase A activity, either by overexpressing negative regulators of the pathway or deleting the upstream effector Ras2, improves the viability of ipl1 and spc24 kinetochore mutants. Ipl1/Aurora B is a highly conserved kinase that corrects attachment of sister kinetochores that have attached to the same spindle pole, whereas Spc24 is a component of the conserved Ndc80 kinetochore complex that attaches directly to microtubules. Unexpectedly, we find that kinetochore mutants have increased phosphorylation levels of protein kinase A substrates, suggesting that the cyclic AMP protein kinase A signaling pathway is stimulated. The increase in protein kinase A activity in kinetochore mutants is not induced by activation of the spindle checkpoint or a metaphase delay because protein kinase A activity remains constant during an unperturbed cell cycle. Finally, we show that lowering protein kinase A activity can rescue the chromosome loss defect of the inner kinetochore ndc10 mutant. Overall, our data suggest that the increased protein kinase A activity in kinetochore mutants is detrimental to cellular growth and chromosome transmission fidelity.

  3. Development of a Split SNAP-CLIP Double Labeling System for Tracking Proteins Following Dissociation from Protein-Protein Complexes in Living Cells.

    PubMed

    Mie, Masayasu; Naoki, Tatsuhiko; Kobatake, Eiry

    2016-08-16

    The split SNAP-tag protein-fragment complementation assay (PCA) is a useful tool for imaging protein-protein interactions (PPIs) in living cells. In contrast to conventional methods employed for imaging PPIs, the split SNAP-tag PCA enables tracking of proteins following dissociation from protein-protein complexes. A limitation of this system, however, is that it only allows for labeling and tracking of one of the proteins forming the protein-protein complex. To track both proteins forming a protein-protein complex, each protein needs to be appropriately labeled. In this study, a split SNAP-CLIP double labeling system is developed and applied for tracking of each protein forming a protein-protein complex. As a proof-of concept, FM protein for PPIs and protein kinase C alpha (PKCα) for translocation are introduced to a split SNAP-CLIP double labeling system. The results show a split SNAP-CLIP double labeling system enables labeling of both proteins in a protein-protein complex and subsequent tracking of each of the proteins following dissociation from the protein-protein complexes in living cells.

  4. Small-Angle X-Ray Scattering From RNA, Proteins, And Protein Complexes

    SciTech Connect

    Lipfert, Jan; Doniach, Sebastian; /Stanford U., Phys. Dept. /Stanford U., Appl. Phys. Dept. /SLAC, SSRL

    2007-09-18

    Small-angle X-ray scattering (SAXS) is increasingly used to characterize the structure and interactions of biological macromolecules and their complexes in solution. Although still a low-resolution technique, the advent of high-flux synchrotron sources and the development of algorithms for the reconstruction of 3-D electron density maps from 1-D scattering profiles have made possible the generation of useful low-resolution molecular models from SAXS data. Furthermore, SAXS is well suited for the study of unfolded or partially folded conformational ensembles as a function of time or solution conditions. Here, we review recently developed algorithms for 3-D structure modeling and applications to protein complexes. Furthermore, we discuss the emerging use of SAXS as a tool to study membrane protein-detergent complexes. SAXS is proving useful to study the folding of functional RNA molecules, and finally we discuss uses of SAXS to study ensembles of denatured proteins.

  5. Modular Broad-Host-Range Expression Vectors for Single-Protein and Protein Complex Purification

    PubMed Central

    Fodor, Barna D.; Kovács, Ákos T.; Csáki, Róbert; Hunyadi-Gulyás, Éva; Klement, Éva; Maróti, Gergely; Mészáros, Lívia S.; Medzihradszky, Katalin F.; Rákhely, Gábor; Kovács, Kornél L.

    2004-01-01

    A set of modular broad-host-range expression vectors with various affinity tags (six-His-tag, FLAG-tag, Strep-tag II, T7-tag) was created. The complete nucleotide sequences of the vectors are known, and these small vectors can be mobilized by conjugation. They are useful in the purification of proteins and protein complexes from gram-negative bacterial species. The plasmids were easily customized for Thiocapsa roseopersicina, Rhodobacter capsulatus, and Methylococcus capsulatus by inserting an appropriate promoter. These examples demonstrate the versatility and flexibility of the vectors. The constructs harbor the T7 promoter for easy overproduction of the desired protein in an appropriate Escherichia coli host. The vectors were useful in purifying different proteins from T. roseopersicina. The FLAG-tag-Strep-tag II combination was utilized for isolation of the HynL-HypC2 protein complex involved in hydrogenase maturation. These tools should be useful for protein purification and for studying protein-protein interactions in a range of bacterial species. PMID:14766546

  6. The synaptonemal complex protein, Zip1, promotes the segregation of nonexchange chromosomes at meiosis I

    PubMed Central

    Newnham, Louise; Jordan, Philip; Rockmill, Beth; Roeder, G. Shirleen; Hoffmann, Eva

    2009-01-01

    Crossing over establishes connections between homologous chromosomes that promote their proper segregation at the first meiotic division. However, there exists a backup system to ensure the correct segregation of those chromosome pairs that fail to cross over. We have found that, in budding yeast, a mutation eliminating the synaptonemal complex protein, Zip1, increases the meiosis I nondisjunction rate of nonexchange chromosomes (NECs). The centromeres of NECs become tethered during meiotic prophase, and this tethering is disrupted by the zip1 mutation. Furthermore, the Zip1 protein often colocalizes to the centromeres of the tethered chromosomes, suggesting that Zip1 plays a direct role in holding NECs together. Zip3, a protein involved in the initiation of synaptonemal complex formation, is also important for NEC segregation. In the absence of Zip3, both the tethering of NECs and the localization of Zip1 to centromeres are impaired. A mutation in the MAD3 gene, which encodes a component of the spindle checkpoint, also increases the nondisjunction of NECs. Together, the zip1 and mad3 mutations have an additive effect, suggesting that these proteins act in parallel pathways to promote NEC segregation. We propose that Mad3 promotes the segregation of NECs that are not tethered by Zip1 at their centromeres. PMID:20080752

  7. Budding Yeast Silencing Complexes and Regulation of Sir2 Activity by Protein-Protein Interactions

    PubMed Central

    Tanny, Jason C.; Kirkpatrick, Donald S.; Gerber, Scott A.; Gygi, Steven P.; Moazed, Danesh

    2004-01-01

    Gene silencing in the budding yeast Saccharomyces cerevisiae requires the enzymatic activity of the Sir2 protein, a highly conserved NAD-dependent deacetylase. In order to study the activity of native Sir2, we purified and characterized two budding yeast Sir2 complexes: the Sir2/Sir4 complex, which mediates silencing at mating-type loci and at telomeres, and the RENT complex, which mediates silencing at the ribosomal DNA repeats. Analyses of the protein compositions of these complexes confirmed previously described interactions. We show that the assembly of Sir2 into native silencing complexes does not alter its selectivity for acetylated substrates, nor does it allow the deacetylation of nucleosomal histones. The inability of Sir2 complexes to deacetylate nucleosomes suggests that additional factors influence Sir2 activity in vivo. In contrast, Sir2 complexes show significant enhancement in their affinities for acetylated substrates and their sensitivities to the physiological inhibitor nicotinamide relative to recombinant Sir2. Reconstitution experiments showed that, for the Sir2/Sir4 complex, these differences stem from the physical interaction of Sir2 with Sir4. Finally, we provide evidence that the different nicotinamide sensitivities of Sir2/Sir4 and RENT in vitro could contribute to locus-specific differences in how Sir2 activity is regulated in vivo. PMID:15282295

  8. Assembly of Neuronal Connectivity by Neurotrophic Factors and Leucine-Rich Repeat Proteins

    PubMed Central

    Ledda, Fernanda; Paratcha, Gustavo

    2016-01-01

    Proper function of the nervous system critically relies on sophisticated neuronal networks interconnected in a highly specific pattern. The architecture of these connections arises from sequential developmental steps such as axonal growth and guidance, dendrite development, target determination, synapse formation and plasticity. Leucine-rich repeat (LRR) transmembrane proteins have been involved in cell-type specific signaling pathways that underlie these developmental processes. The members of this superfamily of proteins execute their functions acting as trans-synaptic cell adhesion molecules involved in target specificity and synapse formation or working in cis as cell-intrinsic modulators of neurotrophic factor receptor trafficking and signaling. In this review, we will focus on novel physiological mechanisms through which LRR proteins regulate neurotrophic factor receptor signaling, highlighting the importance of these modulatory events for proper axonal extension and guidance, tissue innervation and dendrite morphogenesis. Additionally, we discuss few examples linking this set of LRR proteins to neurodevelopmental and psychiatric disorders. PMID:27555809

  9. Assembly of Neuronal Connectivity by Neurotrophic Factors and Leucine-Rich Repeat Proteins.

    PubMed

    Ledda, Fernanda; Paratcha, Gustavo

    2016-01-01

    Proper function of the nervous system critically relies on sophisticated neuronal networks interconnected in a highly specific pattern. The architecture of these connections arises from sequential developmental steps such as axonal growth and guidance, dendrite development, target determination, synapse formation and plasticity. Leucine-rich repeat (LRR) transmembrane proteins have been involved in cell-type specific signaling pathways that underlie these developmental processes. The members of this superfamily of proteins execute their functions acting as trans-synaptic cell adhesion molecules involved in target specificity and synapse formation or working in cis as cell-intrinsic modulators of neurotrophic factor receptor trafficking and signaling. In this review, we will focus on novel physiological mechanisms through which LRR proteins regulate neurotrophic factor receptor signaling, highlighting the importance of these modulatory events for proper axonal extension and guidance, tissue innervation and dendrite morphogenesis. Additionally, we discuss few examples linking this set of LRR proteins to neurodevelopmental and psychiatric disorders. PMID:27555809

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

    USGS Publications Warehouse

    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

  11. Specificity in protein-protein interactions: the structural basis for dual recognition in endonuclease colicin-immunity protein complexes.

    PubMed

    Kühlmann, U C; Pommer, A J; Moore, G R; James, R; Kleanthous, C

    2000-09-01

    Bacteria producing endonuclease colicins are protected against their cytotoxic activity by virtue of a small immunity protein that binds with high affinity and specificity to inactivate the endonuclease. DNase binding by the immunity protein occurs through a "dual recognition" mechanism in which conserved residues from helix III act as the binding-site anchor, while variable residues from helix II define specificity. We now report the 1.7 A crystal structure of the 24.5 kDa complex formed between the endonuclease domain of colicin E9 and its cognate immunity protein Im9, which provides a molecular rationale for this mechanism. Conserved residues of Im9 form a binding-energy hotspot through a combination of backbone hydrogen bonds to the endonuclease, many via buried solvent molecules, and hydrophobic interactions at the core of the interface, while the specificity-determining residues interact with corresponding specificity side-chains on the enzyme. Comparison between the present structure and that reported recently for the colicin E7 endonuclease domain in complex with Im7 highlights how specificity is achieved by very different interactions in the two complexes, predominantly hydrophobic in nature in the E9-Im9 complex but charged in the E7-Im7 complex. A key feature of both complexes is the contact between a conserved tyrosine residue from the immunity proteins (Im9 Tyr54) with a specificity residue on the endonuclease directing it toward the specificity sites of the immunity protein. Remarkably, this tyrosine residue and its neighbour (Im9 Tyr55) are the pivots of a 19 degrees rigid-body rotation that relates the positions of Im7 and Im9 in the two complexes. This rotation does not affect conserved immunity protein interactions with the endonuclease but results in different regions of the specificity helix being presented to the enzyme.

  12. High-resolution diffraction from crystals of a membrane-protein complex: bacterial outer membrane protein OmpC complexed with the antibacterial eukaryotic protein lactoferrin

    SciTech Connect

    Sundara Baalaji, N.; Acharya, K. Ravi; Singh, T. P.; Krishnaswamy, S. E-mail: mkukrishna@rediffmail.com

    2005-08-01

    Crystals of the complex formed between the bacterial membrane protein OmpC and the antibacterial protein lactoferrin suitable for high-resolution structure determination have been obtained. The crystals belong to the hexagonal space group P6, with unit-cell parameters a = b = 116.3, c = 152.4 Å. Crystals of the complex formed between the outer membrane protein OmpC from Escherichia coli and the eukaryotic antibacterial protein lactoferrin from Camelus dromedarius (camel) have been obtained using a detergent environment. Initial data processing suggests that the crystals belong to the hexagonal space group P6, with unit-cell parameters a = b = 116.3, c = 152.4 Å, α = β = 90, γ = 120°. This indicated a Matthews coefficient (V{sub M}) of 3.3 Å{sup 3} Da{sup −1}, corresponding to a possible molecular complex involving four molecules of lactoferrin and two porin trimers in the unit cell (4832 amino acids; 533.8 kDa) with 63% solvent content. A complete set of diffraction data was collected to 3 Å resolution at 100 K. Structure determination by molecular replacement is in progress. Structural study of this first surface-exposed membrane-protein complex with an antibacterial protein will provide insights into the mechanism of action of OmpC as well as lactoferrin.

  13. Contractile vacuole complex--its expanding protein inventory.

    PubMed

    Plattner, Helmut

    2013-01-01

    The contractile vacuole complex (CVC) of some protists serves for the osmotic equilibration of water and ions, notably Ca(2+), by chemiosmotic exploitation of a H(+) gradient generated by the organelle-resident V-type H(+)-ATPase. Ca(2+) is mostly extruded, but there is also some reflux into the cytosol via Ca(2+)-release channels. Most data available are from Dictyostelium and Paramecium. In Paramecium, the major parts of CVC contain several v-/R-SNARE (synaptobrevins) and t-/Q-SNARE (syntaxins) proteins. This is complemented by Rab-type GTPases (shown in Tetrahymena) and exocyst components (Chlamydomonas). All this reflects a multitude of membrane interactions and fusion processes. Ca(2+)/H(+) and other exchangers are to be postulated, as are aquaporins and mechanosensitive Ca(2+) channels. From the complexity of the organelle, many more proteins may be expected. For instance, the pore is endowed with its own set of proteins. We may now envisage the regulation of membrane dynamics (reversible tubulation) and the epigenetic control of organelle shape, size and positioning. New aspects about organelle function and biogenesis are sketched in Section 7. The manifold regulators currently known from CVC suggest the cooperation of widely different mechanisms to maintain its dynamic function and to drive its biogenesis.

  14. Assembly and solution structure of the core retromer protein complex.

    PubMed

    Norwood, Suzanne J; Shaw, Daniel J; Cowieson, Nathan P; Owen, David J; Teasdale, Rohan D; Collins, Brett M

    2011-01-01

    Retromer is a peripheral membrane protein complex that has pleiotropic roles in endosomal membrane trafficking. The core of retromer possesses three subunits, VPS35, VPS29 and VPS26, that play different roles in binding to cargo, regulatory proteins and complex stabilization. We have performed an investigation of the thermodynamics of core retromer assembly using isothermal titration calorimetry (ITC) demonstrating that VPS35 acts as the central subunit to which VPS29 and VPS26 bind independently. Furthermore, we confirm that the conserved PRLYL motif of the large VPS35 subunit is critical for direct VPS26 interaction. Heat capacity measurements of VPS29 and VPS26 binding to VPS35 indicate extensive binding interfaces and suggest conformational alterations in VPS29 or VPS35 upon complex formation. Solution studies of the retromer core using small-angle X-ray scattering allow us to propose a model whereby VPS35 forms an extended platform with VPS29 and VPS26 bound at distal ends, with the potential for forming dimeric assemblies. PMID:20875039

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

    PubMed

    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.

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

    PubMed Central

    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. PMID:26044949

  17. Connectivity between the western and eastern limbs of the Bushveld Complex

    NASA Astrophysics Data System (ADS)

    Cawthorn, R. G.; Webb, S. J.

    2001-01-01

    The mafic layered rocks of the Bushveld Complex are 6-8 km thick and crop out over an area of 65,000 km 2. Previous interpretations of the Bouguer gravity anomalies suggested that the intrusion consisted of two totally separate bodies. However, the mafic sequences in these arcuate western and eastern limbs are remarkably similar, with at least six petrologically distinctive layers and sequences being recognisable in both limbs. Such similarity of sequences in two totally discrete bodies 200-300 km apart is petrologically implausible, and it is suggested that they formed within a single lopolithic intrusion. All previous Bouguer gravity models failed to consider the isostatic response of the crust to emplacement of this huge mass of mafic magma. Isostatic adjustment as a result of this intrusion would have caused the base of the crust to be depressed by as much as 6 km. With this revised whole crustal model, it becomes possible to construct a gravity model, consistent with observed data, which includes a 6 km-thick sequence of mafic rocks connecting the western and eastern limbs of the Bushveld Complex. The exact depth at which the mafic rocks of the Bushveld Complex lie in the centre of the structure cannot be constrained by the gravity data. Such a first-order model is an approximation, because there have been subsequent deformation and structural readjustments in the crust, some of them probably related to the emplacement of the Bushveld Complex. Specifically, the observed geometry of the rocks around the Crocodile River, Dennilton, Marble Hall and Malope Domes suggests that major upwarping of the crust occurred on a variety of scales, triggered by emplacement of the Bushveld Complex.

  18. The CSC connects three major axonemal complexes involved in dynein regulation

    PubMed Central

    Heuser, Thomas; Dymek, Erin E.; Lin, Jianfeng; Smith, Elizabeth F.; Nicastro, Daniela

    2012-01-01

    Motile cilia and flagella are highly conserved organelles that play important roles in human health and development. We recently discovered a calmodulin- and spoke-associ­ated complex (CSC) that is required for wild-type motility and for the stable assembly of a subset of radial spokes. Using cryo–electron tomography, we present the first structure-based localization model of the CSC. Chlamydomonas flagella have two full-length radial spokes, RS1 and RS2, and a shorter RS3 homologue, the RS3 stand-in (RS3S). Using newly developed techniques for analyzing samples with structural heterogeneity, we demonstrate that the CSC connects three major axonemal complexes involved in dynein regulation: RS2, the nexin–dynein regulatory complex (N-DRC), and RS3S. These results provide insights into how signals from the radial spokes may be transmitted to the N-DRC and ultimately to the dynein motors. Our results also indicate that although structurally very similar, RS1 and RS2 likely serve different functions in regulating flagellar motility. PMID:22740634

  19. Young Investigator Award Lecture. Structures of larger proteins, protein-ligand and protein-DNA complexes by multidimensional heteronuclear NMR.

    PubMed Central

    Clore, G. M.; Gronenborn, A. M.

    1994-01-01

    The recent development of a whole panoply of multidimensional heteronuclear-edited and -filtered NMR experiments has revolutionized the field of protein structure determination by NMR, making it possible to extend the methodology from the 10-kDa limit of conventional 2-dimensional NMR to systems up to potentially 35-40 kDa. The basic strategy for solving 3-dimensional structures of larger proteins and protein-ligand complexes in solution using 3- and 4-dimensional NMR spectroscopy is summarized, and the power of these methods is illustrated using 3 examples: interleukin-1 beta, the complex of calmodulin with a target peptide, and the specific complex of the transcription factor GATA-1 with its cognate DNA target site. PMID:8019409

  20. Modeling and fitting protein-protein complexes to predict change of binding energy

    PubMed Central

    Dourado, Daniel F.A.R.; Flores, Samuel Coulbourn

    2016-01-01

    It is possible to accurately and economically predict change in protein-protein interaction energy upon mutation (ΔΔG), when a high-resolution structure of the complex is available. This is of growing usefulness for design of high-affinity or otherwise modified binding proteins for therapeutic, diagnostic, industrial, and basic science applications. Recently the field has begun to pursue ΔΔG prediction for homology modeled complexes, but so far this has worked mostly for cases of high sequence identity. If the interacting proteins have been crystallized in free (uncomplexed) form, in a majority of cases it is possible to find a structurally similar complex which can be used as the basis for template-based modeling. We describe how to use MMB to create such models, and then use them to predict ΔΔG, using a dataset consisting of free target structures, co-crystallized template complexes with sequence identify with respect to the targets as low as 44%, and experimental ΔΔG measurements. We obtain similar results by fitting to a low-resolution Cryo-EM density map. Results suggest that other structural constraints may lead to a similar outcome, making the method even more broadly applicable. PMID:27173910

  1. Targeting protein–protein interactions in complexes organized by A kinase anchoring proteins

    PubMed Central

    Calejo, Ana I.; Taskén, Kjetil

    2015-01-01

    Cyclic AMP is a ubiquitous intracellular second messenger involved in the regulation of a wide variety of cellular processes, a majority of which act through the cAMP – protein kinase A (PKA) signaling pathway and involve PKA phosphorylation of specific substrates. PKA phosphorylation events are typically spatially restricted and temporally well controlled. A-kinase anchoring proteins (AKAPs) directly bind PKA and recruit it to specific subcellular loci targeting the kinase activity toward particular substrates, and thereby provide discrete spatiotemporal control of downstream phosphorylation events. AKAPs also scaffold other signaling molecules into multi-protein complexes that function as crossroads between different signaling pathways. Targeting AKAP coordinated protein complexes with high-affinity peptidomimetics or small molecules to tease apart distinct protein–protein interactions (PPIs) therefore offers important means to disrupt binding of specific components of the complex to better understand the molecular mechanisms involved in the function of individual signalosomes and their pathophysiological role. Furthermore, development of novel classes of small molecules involved in displacement of AKAP-bound signal molecules is now emerging. Here, we will focus on mechanisms for targeting PPI, disruptors that modulate downstream cAMP signaling and their role, especially in the heart. PMID:26441649

  2. GeLC-MS/MS Analysis of Complex Protein Mixtures

    PubMed Central

    Dzieciatkowska, Monika; Hill, Ryan; Hansen, Kirk C.

    2015-01-01

    Discovery-based proteomics has found its place in nearly every facet of biological research. A key objective of this approach is to maximize sequence coverage for proteins across a wide concentration range. Fractionating samples at the protein level is one of the most common ways to circumvent challenges due to sample complexity and improve proteome coverage. Of the available methods, one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by liquid chromatography-tandem mass spectrometry (GeLC-MS/MS) is a robust and reproducible method for qualitative and quantitative proteomic analysis. Here we describe a general GeLC-MS/MS protocol and include technical advice and outline caveats to increase the probability of a successful analysis. PMID:24791981

  3. Force-induced remodelling of proteins and their complexes

    PubMed Central

    Chen, Yun; Radford, Sheena E; Brockwell, David J

    2015-01-01

    Force can drive conformational changes in proteins, as well as modulate their stability and the affinity of their complexes, allowing a mechanical input to be converted into a biochemical output. These properties have been utilised by nature and force is now recognised to be widely used at the cellular level. The effects of force on the biophysical properties of biological systems can be large and varied. As these effects are only apparent in the presence of force, studies on the same proteins using traditional ensemble biophysical methods can yield apparently conflicting results. Where appropriate, therefore, force measurements should be integrated with other experimental approaches to understand the physiological context of the system under study. PMID:25710390

  4. Aberrant Functional Connectivity of the Amygdala Complexes in PTSD during Conscious and Subconscious Processing of Trauma-Related Stimuli.

    PubMed

    Rabellino, Daniela; Densmore, Maria; Frewen, Paul A; Théberge, Jean; McKinnon, Margaret C; Lanius, Ruth A

    2016-01-01

    Post-traumatic stress disorder (PTSD) is characterized by altered functional connectivity of the amygdala complexes at rest. However, amygdala complex connectivity during conscious and subconscious threat processing remains to be elucidated. Here, we investigate specific connectivity of the centromedial amygdala (CMA) and basolateral amygdala (BLA) during conscious and subconscious processing of trauma-related words among individuals with PTSD (n = 26) as compared to non-trauma-exposed controls (n = 20). Psycho-physiological interaction analyses were performed using the right and left amygdala complexes as regions of interest during conscious and subconscious trauma word processing. These analyses revealed a differential, context-dependent responses by each amygdala seed during trauma processing in PTSD. Specifically, relative to controls, during subconscious processing, individuals with PTSD demonstrated increased connectivity of the CMA with the superior frontal gyrus, accompanied by a pattern of decreased connectivity between the BLA and the superior colliculus. During conscious processing, relative to controls, individuals with PTSD showed increased connectivity between the CMA and the pulvinar. These findings demonstrate alterations in amygdala subregion functional connectivity in PTSD and highlight the disruption of the innate alarm network during both conscious and subconscious trauma processing in this disorder. PMID:27631496

  5. Aberrant Functional Connectivity of the Amygdala Complexes in PTSD during Conscious and Subconscious Processing of Trauma-Related Stimuli

    PubMed Central

    Rabellino, Daniela; Densmore, Maria; Frewen, Paul A.; Théberge, Jean; McKinnon, Margaret C.; Lanius, Ruth A.

    2016-01-01

    Post-traumatic stress disorder (PTSD) is characterized by altered functional connectivity of the amygdala complexes at rest. However, amygdala complex connectivity during conscious and subconscious threat processing remains to be elucidated. Here, we investigate specific connectivity of the centromedial amygdala (CMA) and basolateral amygdala (BLA) during conscious and subconscious processing of trauma-related words among individuals with PTSD (n = 26) as compared to non-trauma-exposed controls (n = 20). Psycho-physiological interaction analyses were performed using the right and left amygdala complexes as regions of interest during conscious and subconscious trauma word processing. These analyses revealed a differential, context-dependent responses by each amygdala seed during trauma processing in PTSD. Specifically, relative to controls, during subconscious processing, individuals with PTSD demonstrated increased connectivity of the CMA with the superior frontal gyrus, accompanied by a pattern of decreased connectivity between the BLA and the superior colliculus. During conscious processing, relative to controls, individuals with PTSD showed increased connectivity between the CMA and the pulvinar. These findings demonstrate alterations in amygdala subregion functional connectivity in PTSD and highlight the disruption of the innate alarm network during both conscious and subconscious trauma processing in this disorder. PMID:27631496

  6. Recombinant Expression, Purification, and Functional Characterisation of Connective Tissue Growth Factor and Nephroblastoma-Overexpressed Protein

    PubMed Central

    Bohr, Wilhelm; Kupper, Michael; Hoffmann, Kurt; Weiskirchen, Ralf

    2010-01-01

    The CCN family of proteins, especially its prominent member, the Connective tissue growth factor (CTGF/CCN2) has been identified as a possible biomarker for the diagnosis of fibrotic diseases. As a downstream mediator of TGF-β1 signalling, it is involved in tissue scarring, stimulates interstitial deposition of extracellular matrix proteins, and promotes proliferation of several cell types. Another member of this family, the Nephroblastoma-Overexpressed protein (NOV/CCN3), has growth-inhibiting properties. First reports further suggest that these two CCN family members act opposite to each other in regulating extracellular matrix protein expression and reciprocally influence their own expression when over-expressed. We have established stable HEK and Flp-In-293 clones as productive sources for recombinant human CCN2/CTGF. In addition, we generated an adenoviral vector for recombinant expression of rat NOV and established protocols to purify large quantities of these CCN proteins. The identity of purified human CCN2/CTGF and rat CCN3/NOV was proven by In-gel digest followed by ESI-TOF/MS mass spectrometry. The biological activity of purified proteins was demonstrated using a Smad3-sensitive reporter gene and BrdU proliferation assay in permanent cell line EA•hy 926 cells. We further demonstrate for the first time that both recombinant CCN proteins are N-glycosylated. PMID:21209863

  7. Integration of genomic datasets to predict protein complexes in yeast.

    PubMed

    Jansen, Ronald; Lan, Ning; Qian, Jiang; Gerstein, Mark

    2002-01-01

    The ultimate goal of functional genomics is to define the function of all the genes in the genome of an organism. A large body of information of the biological roles of genes has been accumulated and aggregated in the past decades of research, both from traditional experiments detailing the role of individual genes and proteins, and from newer experimental strategies that aim to characterize gene function on a genomic scale. It is clear that the goal of functional genomics can only be achieved by integrating information and data sources from the variety of these different experiments. Integration of different data is thus an important challenge for bioinformatics. The integration of different data sources often helps to uncover non-obvious relationships between genes, but there are also two further benefits. First, it is likely that whenever information from multiple independent sources agrees, it should be more valid and reliable. Secondly, by looking at the union of multiple sources, one can cover larger parts of the genome. This is obvious for integrating results from multiple single gene or protein experiments, but also necessary for many of the results from genome-wide experiments since they are often confined to certain (although sizable) subsets of the genome. In this paper, we explore an example of such a data integration procedure. We focus on the prediction of membership in protein complexes for individual genes. For this, we recruit six different data sources that include expression profiles, interaction data, essentiality and localization information. Each of these data sources individually contains some weakly predictive information with respect to protein complexes, but we show how this prediction can be improved by combining all of them. Supplementary information is available at http:// bioinfo.mbb.yale.edu/integrate/interactions/. PMID:12836664

  8. The Complex Energy Landscape of the Protein IscU.

    PubMed

    Bothe, Jameson R; Tonelli, Marco; Ali, Ibrahim K; Dai, Ziqi; Frederick, Ronnie O; Westler, William M; Markley, John L

    2015-09-01

    IscU, the scaffold protein for iron-sulfur (Fe-S) cluster biosynthesis in Escherichia coli, traverses a complex energy landscape during Fe-S cluster synthesis and transfer. Our previous studies showed that IscU populates two interconverting conformational states: one structured (S) and one largely disordered (D). Both states appear to be functionally important because proteins involved in the assembly or transfer of Fe-S clusters have been shown to interact preferentially with either the S or D state of IscU. To characterize the complex structure-energy landscape of IscU, we employed NMR spectroscopy, small-angle x-ray scattering (SAXS), and differential scanning calorimetry. Results obtained for IscU at pH 8.0 show that its S state is maximally populated at 25°C and that heating or cooling converts the protein toward the D state. Results from NMR and DSC indicate that both the heat- and cold-induced S→D transitions are cooperative and two-state. Low-resolution structural information from NMR and SAXS suggests that the structures of the cold-induced and heat-induced D states are similar. Both states exhibit similar (1)H-(15)N HSQC spectra and the same pattern of peptidyl-prolyl peptide bond configurations by NMR, and both appear to be similarly expanded compared with the S state based on analysis of SAXS data. Whereas in other proteins the cold-denatured states have been found to be slightly more compact than the heat-denatured states, these two states occupy similar volumes in IscU.

  9. The Complex Energy Landscape of the Protein IscU

    PubMed Central

    Bothe, Jameson R.; Tonelli, Marco; Ali, Ibrahim K.; Dai, Ziqi; Frederick, Ronnie O.; Westler, William M.; Markley, John L.

    2015-01-01

    IscU, the scaffold protein for iron-sulfur (Fe-S) cluster biosynthesis in Escherichia coli, traverses a complex energy landscape during Fe-S cluster synthesis and transfer. Our previous studies showed that IscU populates two interconverting conformational states: one structured (S) and one largely disordered (D). Both states appear to be functionally important because proteins involved in the assembly or transfer of Fe-S clusters have been shown to interact preferentially with either the S or D state of IscU. To characterize the complex structure-energy landscape of IscU, we employed NMR spectroscopy, small-angle x-ray scattering (SAXS), and differential scanning calorimetry. Results obtained for IscU at pH 8.0 show that its S state is maximally populated at 25°C and that heating or cooling converts the protein toward the D state. Results from NMR and DSC indicate that both the heat- and cold-induced S→D transitions are cooperative and two-state. Low-resolution structural information from NMR and SAXS suggests that the structures of the cold-induced and heat-induced D states are similar. Both states exhibit similar 1H-15N HSQC spectra and the same pattern of peptidyl-prolyl peptide bond configurations by NMR, and both appear to be similarly expanded compared with the S state based on analysis of SAXS data. Whereas in other proteins the cold-denatured states have been found to be slightly more compact than the heat-denatured states, these two states occupy similar volumes in IscU. PMID:26331259

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

    NASA Astrophysics Data System (ADS)

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

    2016-05-01

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

  11. A simple stochastic model for complex coextinctions in mutualistic networks: robustness decreases with connectance.

    PubMed

    Vieira, Marcos Costa; Almeida-Neto, Mário

    2015-02-01

    Understanding and predicting species extinctions and coextinctions is a major goal of ecological research in the face of a biodiversity crisis. Typically, models based on network topology are used to simulate coextinctions in mutualistic networks. However, such topological models neglect two key biological features of species interactions: variation in the intrinsic dependence of species on the mutualism, and variation in the relative importance of each interacting partner. By incorporating both types of variation, we developed a stochastic coextinction model capable of simulating extinction cascades far more complex than those observed in previous topological models. Using a set of empirical mutualistic networks, we show that the traditional topological model may either underestimate or overestimate the number and likelihood of coextinctions, depending on the intrinsic dependence of species on the mutualism. More importantly, contrary to topological models, our stochastic model predicts extinction cascades to be more likely in highly connected mutualistic communities. PMID:25431016

  12. Patterns of intercellular connectivity in the mammalian cumulus-oocyte complex.

    PubMed

    Albertini, D F; Rider, V

    1994-02-01

    Electron and fluorescence microscopic techniques have been used in a complementary fashion to study the patterns of follicle cell-oocyte interactions within cumulus-oocyte-complexes of various mammals. The principal findings are: (1) two distinct types of transzonal processes exist that are distinguishable on the basis of cytoskeletal composition; (2) in some of the species examined (pig, goat, primate), corkscrew-shaped processes rich in tubulin, traverse the zona pellucida and are invaginated into the oocyte cortex; (3) actin-rich processes either ramify as a network at the outer surface of the zona pellucida or penetrate the zona and make contact with the oolemma in a species specific manner. These results are discussed with respect both to the need to employ complementary optical methods in assessing connectivity patterns within COC and to the possible role that extracellular matrix-cell interactions play in the homeostatic control of oocyte growth and maturation.

  13. The role of disordered protein regions in the assembly of decapping complexes and RNP granules

    PubMed Central

    Jonas, Stefanie; Izaurralde, Elisa

    2013-01-01

    The removal of the 5′ cap structure by the decapping enzyme DCP2 inhibits translation and generally commits the mRNA to irreversible 5′-to-3′ exonucleolytic degradation by XRN1. DCP2 catalytic activity is stimulated by DCP1, and these proteins form the conserved core of the decapping complex. Additional decapping factors orchestrate the recruitment and activity of this complex in vivo. These factors include enhancer of decapping 3 (EDC3), EDC4, like Sm14A (LSm14A), Pat, the LSm1–7 complex, and the RNA helicase DDX6. Decapping factors are often modular and feature folded domains flanked or connected by low-complexity disordered regions. Recent studies have made important advances in understanding how these disordered regions contribute to the assembly of decapping complexes and promote phase transitions that drive RNP granule formation. These studies have also revealed that the decapping network is governed by interactions mediated by short linear motifs (SLiMs) in these disordered regions. Consequently, the network has rapidly evolved, and although decapping factors are conserved, individual interactions between orthologs have been rewired during evolution. The plasticity of the network facilitates the acquisition of additional subunits or domains in pre-existing subunits, enhances opportunities for regulating mRNA degradation, and eventually leads to the emergence of novel functions. PMID:24352420

  14. Identification of proteins that form specific complexes with the highly conserved protein Translin in Schizosaccharomyces pombe.

    PubMed

    Eliahoo, Elad; Litovco, Phyana; Ben Yosef, Ron; Bendalak, Keren; Ziv, Tamar; Manor, Haim

    2014-04-01

    Translin is a single-stranded DNA and RNA binding protein that has a high affinity for G-rich sequences. TRAX is a Translin paralog that associates with Translin. Both Translin and TRAX were highly conserved in eukaryotes. The nucleic acid binding form of Translin is a barrel-shaped homo-octamer. A Translin-TRAX hetero-octamer having a similar structure also binds nucleic acids. Previous reports suggested that Translin may be involved in chromosomal translocations, telomere metabolism and the control of mRNA transport and translation. More recent studies have indicated that Translin-TRAX hetero-octamers are involved in RNA silencing. To gain a further insight into the functions of Translin, we have undertaken to systematically search for proteins with which it forms specific complexes in living cells. Here we report the results of such a search conducted in the fission yeast Schizosaccharomyces pombe, a suitable model system. This search was carried out by affinity purification and immuno-precipitation techniques, combined with differential labeling of the intracellular proteins with the stable isotopes ¹⁵N and ¹⁴N. We identified for the first time two proteins containing an RNA Recognition Motif (RRM), which are specifically associated with the yeast Translin: (1) the pre-mRNA-splicing factor srp1 that belongs to the highly conserved SR family of proteins and (2) vip1, a protein conserved in fungi. Our data also support the presence of RNA in these intracellular complexes. Our experimental approach should be generally applicable to studies of weak intracellular protein-protein interactions and provides a clear distinction between false positive vs. truly interacting proteins.

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

    PubMed

    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

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

    SciTech Connect

    Nielsen, Anders Lade

    2009-10-23

    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 {gamma}-secretase, a protein protease complex with specificity for intra-membranous cleavage of substrates such as {beta}-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.

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

    PubMed

    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.

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

    PubMed Central

    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

  19. Enhanced conformational sampling to visualize a free-energy landscape of protein complex formation.

    PubMed

    Iida, Shinji; Nakamura, Haruki; Higo, Junichi

    2016-06-15

    We introduce various, recently developed, generalized ensemble methods, which are useful to sample various molecular configurations emerging in the process of protein-protein or protein-ligand binding. The methods introduced here are those that have been or will be applied to biomolecular binding, where the biomolecules are treated as flexible molecules expressed by an all-atom model in an explicit solvent. Sampling produces an ensemble of conformations (snapshots) that are thermodynamically probable at room temperature. Then, projection of those conformations to an abstract low-dimensional space generates a free-energy landscape. As an example, we show a landscape of homo-dimer formation of an endothelin-1-like molecule computed using a generalized ensemble method. The lowest free-energy cluster at room temperature coincided precisely with the experimentally determined complex structure. Two minor clusters were also found in the landscape, which were largely different from the native complex form. Although those clusters were isolated at room temperature, with rising temperature a pathway emerged linking the lowest and second-lowest free-energy clusters, and a further temperature increment connected all the clusters. This exemplifies that the generalized ensemble method is a powerful tool for computing the free-energy landscape, by which one can discuss the thermodynamic stability of clusters and the temperature dependence of the cluster networks.

  20. Enhanced conformational sampling to visualize a free-energy landscape of protein complex formation.

    PubMed

    Iida, Shinji; Nakamura, Haruki; Higo, Junichi

    2016-06-15

    We introduce various, recently developed, generalized ensemble methods, which are useful to sample various molecular configurations emerging in the process of protein-protein or protein-ligand binding. The methods introduced here are those that have been or will be applied to biomolecular binding, where the biomolecules are treated as flexible molecules expressed by an all-atom model in an explicit solvent. Sampling produces an ensemble of conformations (snapshots) that are thermodynamically probable at room temperature. Then, projection of those conformations to an abstract low-dimensional space generates a free-energy landscape. As an example, we show a landscape of homo-dimer formation of an endothelin-1-like molecule computed using a generalized ensemble method. The lowest free-energy cluster at room temperature coincided precisely with the experimentally determined complex structure. Two minor clusters were also found in the landscape, which were largely different from the native complex form. Although those clusters were isolated at room temperature, with rising temperature a pathway emerged linking the lowest and second-lowest free-energy clusters, and a further temperature increment connected all the clusters. This exemplifies that the generalized ensemble method is a powerful tool for computing the free-energy landscape, by which one can discuss the thermodynamic stability of clusters and the temperature dependence of the cluster networks. PMID:27288028

  1. Small Cofactors May Assist Protein Emergence from RNA World: Clues from RNA-Protein Complexes

    PubMed Central

    Shen, Liang; Ji, Hong-Fang

    2011-01-01

    It is now widely accepted that at an early stage in the evolution of life an RNA world arose, in which RNAs both served as the genetic material and catalyzed diverse biochemical reactions. Then, proteins have gradually replaced RNAs because of their superior catalytic properties in catalysis over time. Therefore, it is important to investigate how primitive functional proteins emerged from RNA world, which can shed light on the evolutionary pathway of life from RNA world to the modern world. In this work, we proposed that the emergence of most primitive functional proteins are assisted by the early primitive nucleotide cofactors, while only a minority are induced directly by RNAs based on the analysis of RNA-protein complexes. Furthermore, the present findings have significant implication for exploring the composition of primitive RNA, i.e., adenine base as principal building blocks. PMID:21789260

  2. Characterization of anatomical and functional connectivity in the brain: a complex networks perspective.

    PubMed

    Stam, C J

    2010-09-01

    A central question in modern neuroscience is how anatomical and functional connections between brain areas are organized to allow optimal information processing. In particular, both segregation and integration of information have to be dealt with in a single architecture of brain networks. There is strong evidence that synchronization of neural activity, both locally and between distant regions is a crucial code for functional interactions. However, a powerful theoretical framework to describe the structural and functional topology of system-wide brain networks has only become available with the discovery of 'small-world' and 'scale-free' networks in 1998 and 1999. There is now strong evidence that brain networks, ranging from simple nets of interconnected neurons up to macroscopic networks of brain areas display the typical features of complex systems: high clustering, short path lengths (both typical of 'small-world' networks), skewed degree distributions, presence of hubs, assortative mixing and the presence of modules. This has been demonstrated for anatomical and functional networks using neuroanatomical techniques, EEG, MEG and structural and functional MRI, in organisms ranging from C. elegans to man. In addition, network topology has been shown to be highly heritable, and very predictive of cognitive functioning. A short path length, which implies that from any area in the brain any other area can be reached in a small number of steps, is strongly correlated with IQ. Computational models are now beginning to reveal how the complex structure of adult brain networks could arise during development.

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

    PubMed Central

    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

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

    PubMed

    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

  5. A Repressor Protein Complex Regulates Leaf Growth in Arabidopsis.

    PubMed

    Gonzalez, Nathalie; Pauwels, Laurens; Baekelandt, Alexandra; De Milde, Liesbeth; Van Leene, Jelle; Besbrugge, Nienke; Heyndrickx, Ken S; Cuéllar Pérez, Amparo; Durand, Astrid Nagels; De Clercq, Rebecca; Van De Slijke, Eveline; Vanden Bossche, Robin; Eeckhout, Dominique; Gevaert, Kris; Vandepoele, Klaas; De Jaeger, Geert; Goossens, Alain; Inzé, Dirk

    2015-08-01

    Cell number is an important determinant of final organ size. In the leaf, a large proportion of cells are derived from the stomatal lineage. Meristemoids, which are stem cell-like precursor cells, undergo asymmetric divisions, generating several pavement cells adjacent to the two guard cells. However, the mechanism controlling the asymmetric divisions of these stem cells prior to differentiation is not well understood. Here, we characterized PEAPOD (PPD) proteins, the only transcriptional regulators known to negatively regulate meristemoid division. PPD proteins interact with KIX8 and KIX9, which act as adaptor proteins for the corepressor TOPLESS. D3-type cyclin encoding genes were identified among direct targets of PPD2, being negatively regulated by PPDs and KIX8/9. Accordingly, kix8 kix9 mutants phenocopied PPD loss-of-function producing larger leaves resulting from increased meristemoid amplifying divisions. The identified conserved complex might be specific for leaf growth in the second dimension, since it is not present in Poaceae (grasses), which also lack the developmental program it controls. PMID:26232487

  6. A Repressor Protein Complex Regulates Leaf Growth in Arabidopsis

    PubMed Central

    Gonzalez, Nathalie; Pauwels, Laurens; Baekelandt, Alexandra; De Milde, Liesbeth; Van Leene, Jelle; Besbrugge, Nienke; Heyndrickx, Ken S.; Pérez, Amparo Cuéllar; Durand, Astrid Nagels; De Clercq, Rebecca; Van De Slijke, Eveline; Vanden Bossche, Robin; Eeckhout, Dominique; Gevaert, Kris; Vandepoele, Klaas; De Jaeger, Geert; Goossens, Alain; Inzé, Dirk

    2015-01-01

    Cell number is an important determinant of final organ size. In the leaf, a large proportion of cells are derived from the stomatal lineage. Meristemoids, which are stem cell-like precursor cells, undergo asymmetric divisions, generating several pavement cells adjacent to the two guard cells. However, the mechanism controlling the asymmetric divisions of these stem cells prior to differentiation is not well understood. Here, we characterized PEAPOD (PPD) proteins, the only transcriptional regulators known to negatively regulate meristemoid division. PPD proteins interact with KIX8 and KIX9, which act as adaptor proteins for the corepressor TOPLESS. D3-type cyclin encoding genes were identified among direct targets of PPD2, being negatively regulated by PPDs and KIX8/9. Accordingly, kix8 kix9 mutants phenocopied PPD loss-of-function producing larger leaves resulting from increased meristemoid amplifying divisions. The identified conserved complex might be specific for leaf growth in the second dimension, since it is not present in Poaceae (grasses), which also lack the developmental program it controls. PMID:26232487

  7. Hierarchical structures made of proteins. The complex architecture of spider webs and their constituent silk proteins.

    PubMed

    Heim, Markus; Römer, Lin; Scheibel, Thomas

    2010-01-01

    Biopolymers fulfil a variety of different functions in nature. They conduct various processes inside and outside cells and organisms, with a functionality ranging from storage of information to stabilization, protection, shaping, transport, cellular division, or movement of whole organisms. Within the plethora of biopolymers, the most sophisticated group is of proteinaceous origin: the cytoskeleton of a cell is made of protein filaments that aid in pivotal processes like intracellular transport, movement, and cell division; geckos use a distinct arrangement of keratin-like filaments on their toes which enable them to walk up smooth surfaces, such as walls, and even upside down across ceilings; and spiders spin silks that are extra-corporally used for protection of offspring and construction of complex prey traps. The following tutorial review describes the hierarchical organization of protein fibers, using spider dragline silk as an example. The properties of a dragline silk thread originate from the strictly controlled assembly of the underlying protein chains. The assembly procedure leads to protein fibers showing a complex hierarchical organization comprising three different structural phases. This structural organization is responsible for the outstanding mechanical properties of individual fibers, which out-compete even those of high-performance artificial fibers like Kevlar. Web-weaving spiders produce, in addition to dragline silk, other silks with distinct properties, based on slightly variant constituent proteins--a feature that allows construction of highly sophisticated spider webs with well designed architectures and with optimal mechanical properties for catching prey.

  8. Investigating the subsurface connection beneath Cerro Negro volcano and the El Hoyo Complex, Nicaragua

    NASA Astrophysics Data System (ADS)

    Venugopal, Swetha; Moune, Séverine; Williams-Jones, Glyn

    2016-10-01

    Cerro Negro, the youngest volcano along the Central American Volcanic Belt (CAVB), is a polygenetic cinder cone with relatively frequent basaltic eruptions. The neighbouring El Hoyo complex, of which Las Pilas is the dominant edifice, is a much larger and older complex with milder and less frequent eruptions. Previous studies have suggested a deep link beneath these two closely spaced volcanoes (McKnight, 1995; MacQueen, 2013). Melt inclusions were collected from various tephra samples in order to determine whether a connection exists and to delineate the features of this link. Major, volatile, and trace elemental compositions reveal a distinct geochemical continuum with Cerro Negro defining the primitive endmember and El Hoyo representing the evolved endmember. Magmatic conditions at the time of melt inclusion entrapment were estimated with major and volatile contents: 2.4 kbar and 1170 °C for Cerro Negro melts and 1.3 kbar and 1130 °C for El Hoyo melts with an overall oxygen fugacity at the NNO buffer. Trace element contents are distinct and suggest Cerro Negro magmas fractionally crystallise while El Hoyo magmas are a mix between primitive Cerro Negro melts and residual and evolved El Hoyo magma. Modelling of end member compositions with alphaMELTS confirms the unique nature of El Hoyo magmas as resulting from incremental mixing between Cerro Negro and residual evolved magma at 4 km depth. Combining all available literature data, this study presents a model of the interconnected subsurface plumbing system. This model considers the modern day analogue of the Lemptégy cinder cones in Massif Central, France and incorporates structurally controlled dykes. The main implications of this study are the classification of Cerro Negro as the newest conduit within the El Hoyo Complex as well as the potential re-activation of the El Hoyo edifice.

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

    PubMed

    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.

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

    PubMed

    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

  11. Identifying dynamic protein complexes based on gene expression profiles and PPI networks.

    PubMed

    Li, Min; Chen, Weijie; Wang, Jianxin; Wu, Fang-Xiang; Pan, Yi

    2014-01-01

    Identification of protein complexes from protein-protein interaction networks has become a key problem for understanding cellular life in postgenomic era. Many computational methods have been proposed for identifying protein complexes. Up to now, the existing computational methods are mostly applied on static PPI networks. However, proteins and their interactions are dynamic in reality. Identifying dynamic protein complexes is more meaningful and challenging. In this paper, a novel algorithm, named DPC, is proposed to identify dynamic protein complexes by integrating PPI data and gene expression profiles. According to Core-Attachment assumption, these proteins which are always active in the molecular cycle are regarded as core proteins. The protein-complex cores are identified from these always active proteins by detecting dense subgraphs. Final protein complexes are extended from the protein-complex cores by adding attachments based on a topological character of "closeness" and dynamic meaning. The protein complexes produced by our algorithm DPC contain two parts: static core expressed in all the molecular cycle and dynamic attachments short-lived. The proposed algorithm DPC was applied on the data of Saccharomyces cerevisiae and the experimental results show that DPC outperforms CMC, MCL, SPICi, HC-PIN, COACH, and Core-Attachment based on the validation of matching with known complexes and hF-measures. PMID:24963481

  12. Network reconstruction based on proteomic data and prior knowledge of protein connectivity using graph theory.

    PubMed

    Stavrakas, Vassilis; Melas, Ioannis N; Sakellaropoulos, Theodore; Alexopoulos, Leonidas G

    2015-01-01

    Modeling of signal transduction pathways is instrumental for understanding cells' function. People have been tackling modeling of signaling pathways in order to accurately represent the signaling events inside cells' biochemical microenvironment in a way meaningful for scientists in a biological field. In this article, we propose a method to interrogate such pathways in order to produce cell-specific signaling models. We integrate available prior knowledge of protein connectivity, in a form of a Prior Knowledge Network (PKN) with phosphoproteomic data to construct predictive models of the protein connectivity of the interrogated cell type. Several computational methodologies focusing on pathways' logic modeling using optimization formulations or machine learning algorithms have been published on this front over the past few years. Here, we introduce a light and fast approach that uses a breadth-first traversal of the graph to identify the shortest pathways and score proteins in the PKN, fitting the dependencies extracted from the experimental design. The pathways are then combined through a heuristic formulation to produce a final topology handling inconsistencies between the PKN and the experimental scenarios. Our results show that the algorithm we developed is efficient and accurate for the construction of medium and large scale signaling networks. We demonstrate the applicability of the proposed approach by interrogating a manually curated interaction graph model of EGF/TNFA stimulation against made up experimental data. To avoid the possibility of erroneous predictions, we performed a cross-validation analysis. Finally, we validate that the introduced approach generates predictive topologies, comparable to the ILP formulation. Overall, an efficient approach based on graph theory is presented herein to interrogate protein-protein interaction networks and to provide meaningful biological insights. PMID:26020784

  13. Network reconstruction based on proteomic data and prior knowledge of protein connectivity using graph theory.

    PubMed

    Stavrakas, Vassilis; Melas, Ioannis N; Sakellaropoulos, Theodore; Alexopoulos, Leonidas G

    2015-01-01

    Modeling of signal transduction pathways is instrumental for understanding cells' function. People have been tackling modeling of signaling pathways in order to accurately represent the signaling events inside cells' biochemical microenvironment in a way meaningful for scientists in a biological field. In this article, we propose a method to interrogate such pathways in order to produce cell-specific signaling models. We integrate available prior knowledge of protein connectivity, in a form of a Prior Knowledge Network (PKN) with phosphoproteomic data to construct predictive models of the protein connectivity of the interrogated cell type. Several computational methodologies focusing on pathways' logic modeling using optimization formulations or machine learning algorithms have been published on this front over the past few years. Here, we introduce a light and fast approach that uses a breadth-first traversal of the graph to identify the shortest pathways and score proteins in the PKN, fitting the dependencies extracted from the experimental design. The pathways are then combined through a heuristic formulation to produce a final topology handling inconsistencies between the PKN and the experimental scenarios. Our results show that the algorithm we developed is efficient and accurate for the construction of medium and large scale signaling networks. We demonstrate the applicability of the proposed approach by interrogating a manually curated interaction graph model of EGF/TNFA stimulation against made up experimental data. To avoid the possibility of erroneous predictions, we performed a cross-validation analysis. Finally, we validate that the introduced approach generates predictive topologies, comparable to the ILP formulation. Overall, an efficient approach based on graph theory is presented herein to interrogate protein-protein interaction networks and to provide meaningful biological insights.

  14. Docking and scoring protein complexes: CAPRI 3rd Edition.

    PubMed

    Lensink, Marc F; Méndez, Raúl; Wodak, Shoshana J

    2007-12-01

    The performance of methods for predicting protein-protein interactions at the atomic scale is assessed by evaluating blind predictions performed during 2005-2007 as part of Rounds 6-12 of the community-wide experiment on Critical Assessment of PRedicted Interactions (CAPRI). These Rounds also included a new scoring experiment, where a larger set of models contributed by the predictors was made available to groups developing scoring functions. These groups scored the uploaded set and submitted their own best models for assessment. The structures of nine protein complexes including one homodimer were used as targets. These targets represent biologically relevant interactions involved in gene expression, signal transduction, RNA, or protein processing and membrane maintenance. For all the targets except one, predictions started from the experimentally determined structures of the free (unbound) components or from models derived by homology, making it mandatory for docking methods to model the conformational changes that often accompany association. In total, 63 groups and eight automatic servers, a substantial increase from previous years, submitted docking predictions, of which 1994 were evaluated here. Fifteen groups submitted 305 models for five targets in the scoring experiment. Assessment of the predictions reveals that 31 different groups produced models of acceptable and medium accuracy-but only one high accuracy submission-for all the targets, except the homodimer. In the latter, none of the docking procedures reproduced the large conformational adjustment required for correct assembly, underscoring yet again that handling protein flexibility remains a major challenge. In the scoring experiment, a large fraction of the groups attained the set goal of singling out the correct association modes from incorrect solutions in the limited ensembles of contributed models. But in general they seemed unable to identify the best models, indicating that current scoring

  15. CCM1 and the second life of proteins in adhesion complexes

    PubMed Central

    van den Berg, Maaike CW; Burgering, Boudewijn MT

    2014-01-01

    It is well recognized that a number of proteins present within adhesion complexes perform discrete signaling functions outside these adhesion complexes, including transcriptional control. In this respect, β-catenin is a well-known example of an adhesion protein present both in cadherin complexes and in the nucleus where it regulates the TCF transcription factor. Here we discuss nuclear functions of adhesion complex proteins with a special focus on the CCM-1/KRIT-1 protein, which may turn out to be yet another adhesion complex protein with a second life. PMID:24714220

  16. Silencio/CG9754 connects the Piwi-piRNA complex to the cellular heterochromatin machinery.

    PubMed

    Sienski, Grzegorz; Batki, Julia; Senti, Kirsten-André; Dönertas, Derya; Tirian, Laszlo; Meixner, Katharina; Brennecke, Julius

    2015-11-01

    The repression of transposable elements in eukaryotes often involves their transcriptional silencing via targeted chromatin modifications. In animal gonads, nuclear Argonaute proteins of the PIWI clade complexed with small guide RNAs (piRNAs) serve as sequence specificity determinants in this process. How binding of nuclear PIWI-piRNA complexes to nascent transcripts orchestrates heterochromatin formation and transcriptional silencing is unknown. Here, we characterize CG9754/Silencio as an essential piRNA pathway factor that is required for Piwi-mediated transcriptional silencing in Drosophila. Ectopic targeting of Silencio to RNA or DNA is sufficient to elicit silencing independently of Piwi and known piRNA pathway factors. Instead, Silencio requires the H3K9 methyltransferase Eggless/SetDB1 for its silencing ability. In agreement with this, SetDB1, but not Su(var)3-9, is required for Piwi-mediated transcriptional silencing genome-wide. Due to its interaction with the target-engaged Piwi-piRNA complex, we suggest that Silencio acts as linker between the sequence specificity factor Piwi and the cellular heterochromatin machinery.

  17. Silencio/CG9754 connects the Piwi–piRNA complex to the cellular heterochromatin machinery

    PubMed Central

    Sienski, Grzegorz; Batki, Julia; Senti, Kirsten-André; Dönertas, Derya; Tirian, Laszlo; Meixner, Katharina; Brennecke, Julius

    2015-01-01

    The repression of transposable elements in eukaryotes often involves their transcriptional silencing via targeted chromatin modifications. In animal gonads, nuclear Argonaute proteins of the PIWI clade complexed with small guide RNAs (piRNAs) serve as sequence specificity determinants in this process. How binding of nuclear PIWI–piRNA complexes to nascent transcripts orchestrates heterochromatin formation and transcriptional silencing is unknown. Here, we characterize CG9754/Silencio as an essential piRNA pathway factor that is required for Piwi-mediated transcriptional silencing in Drosophila. Ectopic targeting of Silencio to RNA or DNA is sufficient to elicit silencing independently of Piwi and known piRNA pathway factors. Instead, Silencio requires the H3K9 methyltransferase Eggless/SetDB1 for its silencing ability. In agreement with this, SetDB1, but not Su(var)3-9, is required for Piwi-mediated transcriptional silencing genome-wide. Due to its interaction with the target-engaged Piwi–piRNA complex, we suggest that Silencio acts as linker between the sequence specificity factor Piwi and the cellular heterochromatin machinery. PMID:26494711

  18. Quantitative characterization of protein-protein complexes involved in base excision DNA repair.

    PubMed

    Moor, Nina A; Vasil'eva, Inna A; Anarbaev, Rashid O; Antson, Alfred A; Lavrik, Olga I

    2015-07-13

    Base Excision Repair (BER) efficiently corrects the most common types of DNA damage in mammalian cells. Step-by-step coordination of BER is facilitated by multiple interactions between enzymes and accessory proteins involved. Here we characterize quantitatively a number of complexes formed by DNA polymerase β (Polβ), apurinic/apyrimidinic endonuclease 1 (APE1), poly(ADP-ribose) polymerase 1 (PARP1), X-ray repair cross-complementing protein 1 (XRCC1) and tyrosyl-DNA phosphodiesterase 1 (TDP1), using fluorescence- and light scattering-based techniques. Direct physical interactions between the APE1-Polβ, APE1-TDP1, APE1-PARP1 and Polβ-TDP1 pairs have been detected and characterized for the first time. The combined results provide strong evidence that the most stable complex is formed between XRCC1 and Polβ. Model DNA intermediates of BER are shown to induce significant rearrangement of the Polβ complexes with XRCC1 and PARP1, while having no detectable influence on the protein-protein binding affinities. The strength of APE1 interaction with Polβ, XRCC1 and PARP1 is revealed to be modulated by BER intermediates to different extents, depending on the type of DNA damage. The affinity of APE1 for Polβ is higher in the complex with abasic site-containing DNA than after the APE1-catalyzed incision. Our findings advance understanding of the molecular mechanisms underlying coordination and regulation of the BER process.

  19. Heterodimeric protein complex identification by naïve Bayes classifiers

    PubMed Central

    2013-01-01

    Background Protein complexes are basic cellular entities that carry out the functions of their components. It can be found that in databases of protein complexes of yeast like CYC2008, the major type of known protein complexes is heterodimeric complexes. Although a number of methods for trying to predict sets of proteins that form arbitrary types of protein complexes simultaneously have been proposed, it can be found that they often fail to predict heterodimeric complexes. Results In this paper, we have designed several features characterizing heterodimeric protein complexes based on genomic data sets, and proposed a supervised-learning method for the prediction of heterodimeric protein complexes. This method learns the parameters of the features, which are embedded in the naïve Bayes classifier. The log-likelihood ratio derived from the naïve Bayes classifier with the parameter values obtained by maximum likelihood estimation gives the score of a given pair of proteins to predict whether the pair is a heterodimeric complex or not. A five-fold cross-validation shows good performance on yeast. The trained classifiers also show higher predictability than various existing algorithms on yeast data sets with approximate and exact matching criteria. Conclusions Heterodimeric protein complex prediction is a rather harder problem than heteromeric protein complex prediction because heterodimeric protein complex is topologically simpler. However, it turns out that by designing features specialized for heterodimeric protein complexes, predictability of them can be improved. Thus, the design of more sophisticate features for heterodimeric protein complexes as well as the accumulation of more accurate and useful genome-wide data sets will lead to higher predictability of heterodimeric protein complexes. Our tool can be downloaded from http://imi.kyushu-u.ac.jp/~om/. PMID:24299017

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

    PubMed Central

    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

  1. RECOVERY ACT - Robust Optimization for Connectivity and Flows in Dynamic Complex Networks

    SciTech Connect

    Balasundaram, Balabhaskar; Butenko, Sergiy; Boginski, Vladimir; Uryasev, Stan

    2013-12-25

    The goal of this project was to study robust connectivity and flow patterns of complex multi-scale systems modeled as networks. Networks provide effective ways to study global, system level properties, as well as local, multi-scale interactions at a component level. Numerous applications from power systems, telecommunication, transportation, biology, social science, and other areas have benefited from novel network-based models and their analysis. Modeling and optimization techniques that employ appropriate measures of risk for identifying robust clusters and resilient network designs in networks subject to uncertain failures were investigated in this collaborative multi-university project. In many practical situations one has to deal with uncertainties associated with possible failures of network components, thereby affecting the overall efficiency and performance of the system (e.g., every node/connection has a probability of partial or complete failure). Some extreme examples include power grid component failures, airline hub failures due to weather, or freeway closures due to emergencies. These are also situations in which people, materials, or other resources need to be managed efficiently. Important practical examples include rerouting flow through power grids, adjusting flight plans, and identifying routes for emergency services and supplies, in the event network elements fail unexpectedly. Solutions that are robust under uncertainty, in addition to being economically efficient, are needed. This project has led to the development of novel models and methodologies that can tackle the optimization problems arising in such situations. A number of new concepts, which have not been previously applied in this setting, were investigated in the framework of the project. The results can potentially help decision-makers to better control and identify robust or risk-averse decisions in such situations. Formulations and optimal solutions of the considered problems need

  2. Atomistic Simulation of Lignocellulosic Biomass and Associated Cellulosomal Protein Complexes

    SciTech Connect

    Petridis, Loukas; Crowley, Michael F; Smith, Jeremy C

    2010-01-01

    Computer simulations have been performed to obtain an atomic-level understanding of lignocellulose structure and the assembly of its associated cellulosomal protein complexes. First, a CHARMM molecular mechanics force field for lignin is derived and validated by performing a molecular dynamics simulation of a crystal of a lignin fragment molecule and comparing simulation-derived structural features with experimental results. Together with the existing force field for polysaccharides, this work provides the basis for full simulations of lignocellulose. Second, the underlying molecular mechanism governing the assembly of various cellulosomal modules is investigated by performing a novel free-energy calculation of the cohesin-dockerin dissociation. Our calculation indicates a free-energy barrier of ~17 kcal/mol and further reveals a stepwise dissociation pathway involving both the central -sheet interface and its adjacent solvent-exposed loop/turn regions clustered at both ends of the -barrel structure.

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

    PubMed Central

    Dygut, Jacek; Kalinowska, Barbara; Banach, Mateusz; Piwowar, Monika; Konieczny, Leszek; Roterman, Irena

    2016-01-01

    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. PMID:27763556

  4. Free energy landscapes of encounter complexes in protein-protein association.

    PubMed Central

    Camacho, C J; Weng, Z; Vajda, S; DeLisi, C

    1999-01-01

    We report the computer generation of a high-density map of the thermodynamic properties of the diffusion-accessible encounter conformations of four receptor-ligand protein pairs, and use it to study the electrostatic and desolvation components of the free energy of association. Encounter complex conformations are generated by sampling the translational/rotational space of the ligand around the receptor, both at 5-A and zero surface-to-surface separations. We find that partial desolvation is always an important effect, and it becomes dominant for complexes in which one of the reactants is neutral or weakly charged. The interaction provides a slowly varying attractive force over a small but significant region of the molecular surface. In complexes with no strong charge complementarity this region surrounds the binding site, and the orientation of the ligand in the encounter conformation with the lowest desolvation free energy is similar to the one observed in the fully formed complex. Complexes with strong opposite charges exhibit two types of behavior. In the first group, represented by barnase/barstar, electrostatics exerts strong orientational steering toward the binding site, and desolvation provides some added adhesion within the local region of low electrostatic energy. In the second group, represented by the complex of kallikrein and pancreatic trypsin inhibitor, the overall stability results from the rather nonspecific electrostatic attraction, whereas the affinity toward the binding region is determined by desolvation interactions. PMID:10049302

  5. RNA and protein complexes of trp RNA-binding attenuation protein characterized by mass spectrometry.

    PubMed

    Akashi, Satoko; Watanabe, Masahiro; Heddle, Jonathan G; Unzai, Satoru; Park, Sam-Yong; Tame, Jeremy R H

    2009-03-15

    We have characterized both wild-type and mutant TRAP (trp RNA-binding attenuation protein) from Bacillus stearothermophilus , and their complexes with RNA or its regulator anti-TRAP protein (AT), by electrospray ionization mass spectrometry (ESI-MS). Wild-type TRAP mainly 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 polypeptide molecules. Mass spectra showed that both the wild-type TRAP 11mer and the mutant TRAP 12mer can bind a cognate single-stranded RNA molecule with a molar ratio of 1:1. The crystal structure of wild-type TRAP complexed with AT shows a TRAP 12mer ring surrounded by six AT trimers. However, nanoESI-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. These results suggest that crystallization selects only the most symmetrical TRAP-AT complex from the solution, whereas ESI-MS can take a "snapshot" of all the species in solution. PMID:19219981

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

    PubMed

    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

  7. Robustness, efficiency, and optimality in the Fenna-Matthews-Olson photosynthetic pigment-protein complex

    NASA Astrophysics Data System (ADS)

    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

  8. Robustness, efficiency, and optimality in the Fenna-Matthews-Olson photosynthetic pigment-protein complex

    SciTech Connect

    Baker, Lewis A.; Habershon, Scott

    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

  9. Complex interactions of HIV-1 nucleocapsid protein with oligonucleotides.

    PubMed

    Fisher, Robert J; Fivash, Matthew J; Stephen, Andrew G; Hagan, Nathan A; Shenoy, Shilpa R; Medaglia, Maxine V; Smith, Lindsey R; Worthy, Karen M; Simpson, John T; Shoemaker, Robert; McNitt, Karen L; Johnson, Donald G; Hixson, Catherine V; Gorelick, Robert J; Fabris, Daniele; Henderson, Louis E; Rein, Alan

    2006-01-01

    The HIV-1 nucleocapsid (NC) protein is a small, basic protein containing two retroviral zinc fingers. It is a highly active nucleic acid chaperone; because of this activity, it plays a crucial role in virus replication as a cofactor during reverse transcription, and is probably important in other steps of the replication cycle as well. We previously reported that NC binds with high-affinity to the repeating sequence d(TG)n. We have now analyzed the interaction between NC and d(TG)4 in considerable detail, using surface plasmon resonance (SPR), tryptophan fluorescence quenching (TFQ), fluorescence anisotropy (FA), isothermal titration calorimetry (ITC) and electrospray ionization Fourier transform mass spectrometry (ESI-FTMS). Our results show that the interactions between these two molecules are surprisngly complex: while the K(d) for binding of a single d(TG)4 molecule to NC is only approximately 5 nM in 150 mM NaCl, a single NC molecule is capable of interacting with more than one d(TG)4 molecule, and conversely, more than one NC molecule can bind to a single d(TG)4 molecule. The strengths of these additional binding reactions are quantitated. The implications of this multivalency for the functions of NC in virus replication are discussed.

  10. Cell-surface Attachment of Bacterial Multienzyme Complexes Involves Highly Dynamic Protein-Protein Anchors*

    PubMed Central

    Cameron, Kate; Najmudin, Shabir; Alves, Victor D.; Bayer, Edward A.; Smith, Steven P.; Bule, Pedro; Waller, Helen; Ferreira, Luís M. A.; Gilbert, Harry J.; Fontes, Carlos M. G. A.

    2015-01-01

    Protein-protein interactions play a pivotal role in the assembly of the cellulosome, one of nature's most intricate nanomachines dedicated to the depolymerization of complex carbohydrates. The integration of cellulosomal components usually occurs through the binding of type I dockerin modules located at the C terminus of the enzymes to cohesin modules located in the primary scaffoldin subunit. Cellulosomes are typically recruited to the cell surface via type II cohesin-dockerin interactions established between primary and cell-surface anchoring scaffoldin subunits. In contrast with type II interactions, type I dockerins usually display a dual binding mode that may allow increased conformational flexibility during cellulosome assembly. Acetivibrio cellulolyticus produces a highly complex cellulosome comprising an unusual adaptor scaffoldin, ScaB, which mediates the interaction between the primary scaffoldin, ScaA, through type II cohesin-dockerin interactions and the anchoring scaffoldin, ScaC, via type I cohesin-dockerin interactions. Here, we report the crystal structure of the type I ScaB dockerin in complex with a type I ScaC cohesin in two distinct orientations. The data show that the ScaB dockerin displays structural symmetry, reflected by the presence of two essentially identical binding surfaces. The complex interface is more extensive than those observed in other type I complexes, which results in an ultra-high affinity interaction (Ka ∼1012 m). A subset of ScaB dockerin residues was also identified as modulating the specificity of type I cohesin-dockerin interactions in A. cellulolyticus. This report reveals that recruitment of cellulosomes onto the cell surface may involve dockerins presenting a dual binding mode to incorporate additional flexibility into the quaternary structure of highly populated multienzyme complexes. PMID:25855788

  11. Identification of human protein complexes from local sub-graphs of protein-protein interaction network based on random forest with topological structure features.

    PubMed

    Li, Zhan-Chao; Lai, Yan-Hua; Chen, Li-Li; Zhou, Xuan; Dai, Zong; Zou, Xiao-Yong

    2012-03-01

    In the post-genomic era, one of the most important and challenging tasks is to identify protein complexes and further elucidate its molecular mechanisms in specific biological processes. Previous computational approaches usually identify protein complexes from protein interaction network based on dense sub-graphs and incomplete priori information. Additionally, the computational approaches have little concern about the biological properties of proteins and there is no a common evaluation metric to evaluate the performance. So, it is necessary to construct novel method for identifying protein complexes and elucidating the function of protein complexes. In this study, a novel approach is proposed to identify protein complexes using random forest and topological structure. Each protein complex is represented by a graph of interactions, where descriptor of the protein primary structure is used to characterize biological properties of protein and vertex is weighted by the descriptor. The topological structure features are developed and used to characterize protein complexes. Random forest algorithm is utilized to build prediction model and identify protein complexes from local sub-graphs instead of dense sub-graphs. As a demonstration, the proposed approach is applied to protein interaction data in human, and the satisfied results are obtained with accuracy of 80.24%, sensitivity of 81.94%, specificity of 80.07%, and Matthew's correlation coefficient of 0.4087 in 10-fold cross-validation test. Some new protein complexes are identified, and analysis based on Gene Ontology shows that the complexes are likely to be true complexes and play important roles in the pathogenesis of some diseases. PCI-RFTS, a corresponding executable program for protein complexes identification, can be acquired freely on request from the authors.

  12. The yeast p24 complex regulates GPI-anchored protein transport and quality control by monitoring anchor remodeling

    PubMed Central

    Castillon, Guillaume A.; Aguilera-Romero, Auxiliadora; Manzano-Lopez, Javier; Epstein, Sharon; Kajiwara, Kentaro; Funato, Kouichi; Watanabe, Reika; Riezman, Howard; Muñiz, Manuel

    2011-01-01

    Glycosylphosphatidylinositol (GPI)-anchored proteins are secretory proteins that are attached to the cell surface of eukaryotic cells by a glycolipid moiety. Once GPI anchoring has occurred in the lumen of the endoplasmic reticulum (ER), the structure of the lipid part on the GPI anchor undergoes a remodeling process prior to ER exit. In this study, we provide evidence suggesting that the yeast p24 complex, through binding specifically to GPI-anchored proteins in an anchor-dependent manner, plays a dual role in their selective trafficking. First, the p24 complex promotes efficient ER exit of remodeled GPI-anchored proteins after concentration by connecting them with the COPII coat and thus facilitates their incorporation into vesicles. Second, it retrieves escaped, unremodeled GPI-anchored proteins from the Golgi to the ER in COPI vesicles. Therefore the p24 complex, by sensing the status of the GPI anchor, regulates GPI-anchored protein intracellular transport and coordinates this with correct anchor remodeling. PMID:21680708

  13. Structural basis for translational surveillance by the large ribosomal subunit-associated protein quality control complex

    PubMed Central

    Lyumkis, Dmitry; Oliveira dos Passos, Dario; Tahara, Erich B.; Webb, Kristofor; Bennett, Eric J.; Vinterbo, Staal; Potter, Clinton S.; Carragher, Bridget; Joazeiro, Claudio A. P.

    2014-01-01

    All organisms have evolved mechanisms to manage the stalling of ribosomes upon translation of aberrant mRNA. In eukaryotes, the large ribosomal subunit-associated quality control complex (RQC), composed of the listerin/Ltn1 E3 ubiquitin ligase and cofactors, mediates the ubiquitylation and extraction of ribosome-stalled nascent polypeptide chains for proteasomal degradation. How RQC recognizes stalled ribosomes and performs its functions has not been understood. Using single-particle cryoelectron microscopy, we have determined the structure of the RQC complex bound to stalled 60S ribosomal subunits. The structure establishes how Ltn1 associates with the large ribosomal subunit and properly positions its E3-catalytic RING domain to mediate nascent chain ubiquitylation. The structure also reveals that a distinguishing feature of stalled 60S particles is an exposed, nascent chain-conjugated tRNA, and that the Tae2 subunit of RQC, which facilitates Ltn1 binding, is responsible for selective recognition of stalled 60S subunits. RQC components are engaged in interactions across a large span of the 60S subunit surface, connecting the tRNA in the peptidyl transferase center to the distally located nascent chain tunnel exit. This work provides insights into a mechanism linking translation and protein degradation that targets defective proteins immediately after synthesis, while ignoring nascent chains in normally translating ribosomes. PMID:25349383

  14. Septal complex of the telencephalon of lizards: III. Efferent connections and general discussion.

    PubMed

    Font, C; Lanuza, E; Martinez-Marcos, A; Hoogland, P V; Martinez-Garcia, F

    1998-11-30

    The projections of the septum of the lizard Podarcis hispanica (Lacertidae) were studied by combining retrograde and anterograde neuroanatomical tracing. The results confirm the classification of septal nuclei into three main divisions. The nuclei composing the central septal division (anterior, lateral, medial, dorsolateral, and ventrolateral nuclei) displayed differential projections to the basal telencephalon, preoptic and anterior hypothalamus, lateral hypothalamic area, dorsal hypothalamus, mammillary complex, dorsomedial anterior thalamus, ventral tegmental area, interpeduncular nucleus, raphe nucleus, torus semicircularis pars laminaris, reptilian A8 nucleus/substantia nigra and central gray. For instance, only the medial septal nucleus projected substantially to the thalamus whereas the anterior septum was the only nucleus projecting to the caudal midbrain including the central gray. The anterior and lateral septal nuclei also differ in the way in which their projection to the preoptic hypothalamus terminated. The midline septal division is composed of the dorsal septal nucleus, nucleus septalis impar and nucleus of the posterior pallial commissure. The latter two nuclei projected to the lateral habenula and, at least the nucleus of the posterior pallial commissure, to the mammillary complex. The dorsal septal nucleus projected to the preoptic and periventricular hypothalamus and the anterior thalamus, but its central part seemed to project to the caudal midbrain (up to the midbrain central gray). Finally, the ventromedial septal division (ventromedial septal nucleus) showed a massive projection to the anterior and the lateral tuberomammillary hypothalamus. Data on the connections of the septum of P. hispanica and Gecko gekko are discussed from a comparative point of view and used for better understanding of the functional anatomy of the tetrapodian septum.

  15. Hierarchical organization of functional connectivity in the mouse brain: a complex network approach

    NASA Astrophysics Data System (ADS)

    Bardella, Giampiero; Bifone, Angelo; Gabrielli, Andrea; Gozzi, Alessandro; Squartini, Tiziano

    2016-08-01

    This paper represents a contribution to the study of the brain functional connectivity from the perspective of complex networks theory. More specifically, we apply graph theoretical analyses to provide evidence of the modular structure of the mouse brain and to shed light on its hierarchical organization. We propose a novel percolation analysis and we apply our approach to the analysis of a resting-state functional MRI data set from 41 mice. This approach reveals a robust hierarchical structure of modules persistent across different subjects. Importantly, we test this approach against a statistical benchmark (or null model) which constrains only the distributions of empirical correlations. Our results unambiguously show that the hierarchical character of the mouse brain modular structure is not trivially encoded into this lower-order constraint. Finally, we investigate the modular structure of the mouse brain by computing the Minimal Spanning Forest, a technique that identifies subnetworks characterized by the strongest internal correlations. This approach represents a faster alternative to other community detection methods and provides a means to rank modules on the basis of the strength of their internal edges.

  16. Hierarchical organization of functional connectivity in the mouse brain: a complex network approach.

    PubMed

    Bardella, Giampiero; Bifone, Angelo; Gabrielli, Andrea; Gozzi, Alessandro; Squartini, Tiziano

    2016-01-01

    This paper represents a contribution to the study of the brain functional connectivity from the perspective of complex networks theory. More specifically, we apply graph theoretical analyses to provide evidence of the modular structure of the mouse brain and to shed light on its hierarchical organization. We propose a novel percolation analysis and we apply our approach to the analysis of a resting-state functional MRI data set from 41 mice. This approach reveals a robust hierarchical structure of modules persistent across different subjects. Importantly, we test this approach against a statistical benchmark (or null model) which constrains only the distributions of empirical correlations. Our results unambiguously show that the hierarchical character of the mouse brain modular structure is not trivially encoded into this lower-order constraint. Finally, we investigate the modular structure of the mouse brain by computing the Minimal Spanning Forest, a technique that identifies subnetworks characterized by the strongest internal correlations. This approach represents a faster alternative to other community detection methods and provides a means to rank modules on the basis of the strength of their internal edges. PMID:27534708

  17. Hierarchical organization of functional connectivity in the mouse brain: a complex network approach

    PubMed Central

    Bardella, Giampiero; Bifone, Angelo; Gabrielli, Andrea; Gozzi, Alessandro; Squartini, Tiziano

    2016-01-01

    This paper represents a contribution to the study of the brain functional connectivity from the perspective of complex networks theory. More specifically, we apply graph theoretical analyses to provide evidence of the modular structure of the mouse brain and to shed light on its hierarchical organization. We propose a novel percolation analysis and we apply our approach to the analysis of a resting-state functional MRI data set from 41 mice. This approach reveals a robust hierarchical structure of modules persistent across different subjects. Importantly, we test this approach against a statistical benchmark (or null model) which constrains only the distributions of empirical correlations. Our results unambiguously show that the hierarchical character of the mouse brain modular structure is not trivially encoded into this lower-order constraint. Finally, we investigate the modular structure of the mouse brain by computing the Minimal Spanning Forest, a technique that identifies subnetworks characterized by the strongest internal correlations. This approach represents a faster alternative to other community detection methods and provides a means to rank modules on the basis of the strength of their internal edges. PMID:27534708

  18. Disulfide connectivity and reduction in pheromone-binding proteins of the gypsy moth, Lymantria dispar

    NASA Astrophysics Data System (ADS)

    Honson, Nicolette S.; Plettner, Erika

    2006-06-01

    Males of the gypsy moth, Lymantria dispar, are attracted by a pheromone released by females. Pheromones are detected by olfactory neurons housed in specialized sensory hairs located on the antennae of the male moth. Once pheromone molecules enter the sensilla lymph, a highly abundant pheromone-binding protein (PBP) transports the molecule to the sensory neuron. The PBPs are members of the insect odorant-binding protein family, with six conserved cysteine residues. In this study, the disulfide bond connectivities of the pheromone-binding proteins PBP1 and PBP2 from the gypsy moth were found to be cysteines 19-54, 50-109, and 97-118 for PBP1, and cysteines 19-54, 50-110, and 97-119 for PBP2, as determined by cyanylation reactions and cyanogen bromide chemical cleavage. We have discovered that the second disulfide linkage is the most easily reduced of the three, and this same linkage is missing among four cysteine-containing insect odorant-binding proteins (OBPs). We are the first to identify the unique steric and electronic properties of this second disulfide linkage.

  19. Disulfide connectivity and reduction in pheromone-binding proteins of the gypsy moth, Lymantria dispar.

    PubMed

    Honson, Nicolette S; Plettner, Erika

    2006-06-01

    Males of the gypsy moth, Lymantria dispar, are attracted by a pheromone released by females. Pheromones are detected by olfactory neurons housed in specialized sensory hairs located on the antennae of the male moth. Once pheromone molecules enter the sensilla lymph, a highly abundant pheromone-binding protein (PBP) transports the molecule to the sensory neuron. The PBPs are members of the insect odorant-binding protein family, with six conserved cysteine residues. In this study, the disulfide bond connectivities of the pheromone-binding proteins PBP1 and PBP2 from the gypsy moth were found to be cysteines 19-54, 50-109, and 97-118 for PBP1, and cysteines 19-54, 50-110, and 97-119 for PBP2, as determined by cyanylation reactions and cyanogen bromide chemical cleavage. We have discovered that the second disulfide linkage is the most easily reduced of the three, and this same linkage is missing among four cysteine-containing insect odorant-binding proteins (OBPs). We are the first to identify the unique steric and electronic properties of this second disulfide linkage.

  20. Structures of neuroligin-1 and the neuroligin-1/neurexin-1 beta complex reveal specific protein-protein and protein-Ca2+ interactions.

    PubMed

    Araç, Demet; Boucard, Antony A; Ozkan, Engin; Strop, Pavel; Newell, Evan; Südhof, Thomas C; Brunger, Axel T

    2007-12-20

    Neurexins and neuroligins provide trans-synaptic connectivity by the Ca2+-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 Ca2+. 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.

  1. Structures of Neuroligin-1 And the Neuroligin-1/Neurexin-1 Beta Complex Reveal Specific Protein-Protein And Protein-Ca2+ Interactions

    SciTech Connect

    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.

  2. Structures of Neuroligin-1 And the Neuroligin-1/Neurexin-1beta Complex Reveal Specific Protein-Protein And Protein-Ca**2+ Interactions

    SciTech Connect

    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.

  3. Functional Mapping of Protein-Protein Interactions in an Enzyme Complex by Directed Evolution

    PubMed Central

    Roderer, Kathrin; Neuenschwander, Martin; Codoni, Giosiana; Sasso, Severin; Gamper, Marianne; Kast, Peter

    2014-01-01

    The shikimate pathway enzyme chorismate mutase converts chorismate into prephenate, a precursor of Tyr and Phe. The intracellular chorismate mutase (MtCM) of Mycobacterium tuberculosis is poorly active on its own, but becomes >100-fold more efficient upon formation of a complex with the first enzyme of the shikimate pathway, 3-deoxy-d-arabino-heptulosonate-7-phosphate synthase (MtDS). The crystal structure of the enzyme complex revealed involvement of C-terminal MtCM residues with the MtDS interface. Here we employed evolutionary strategies to probe the tolerance to substitution of the C-terminal MtCM residues from positions 84–90. Variants with randomized positions were subjected to stringent selection in vivo requiring productive interactions with MtDS for survival. Sequence patterns identified in active library members coincide with residue conservation in natural chorismate mutases of the AroQδ subclass to which MtCM belongs. An Arg-Gly dyad at positions 85 and 86, invariant in AroQδ sequences, was intolerant to mutation, whereas Leu88 and Gly89 exhibited a preference for small and hydrophobic residues in functional MtCM-MtDS complexes. In the absence of MtDS, selection under relaxed conditions identifies positions 84–86 as MtCM integrity determinants, suggesting that the more C-terminal residues function in the activation by MtDS. Several MtCM variants, purified using a novel plasmid-based T7 RNA polymerase gene expression system, showed that a diminished ability to physically interact with MtDS correlates with reduced activatability and feedback regulatory control by Tyr and Phe. Mapping critical protein-protein interaction sites by evolutionary strategies may pinpoint promising targets for drugs that interfere with the activity of protein complexes. PMID:25551646

  4. MOEPGA: A novel method to detect protein complexes in yeast protein-protein interaction networks based on MultiObjective Evolutionary Programming Genetic Algorithm.

    PubMed

    Cao, Buwen; Luo, Jiawei; Liang, Cheng; Wang, Shulin; Song, Dan

    2015-10-01

    The identification of protein complexes in protein-protein interaction (PPI) networks has greatly advanced our understanding of biological organisms. Existing computational methods to detect protein complexes are usually based on specific network topological properties of PPI networks. However, due to the inherent complexity of the network structures, the identification of protein complexes may not be fully addressed by using single network topological property. In this study, we propose a novel MultiObjective Evolutionary Programming Genetic Algorithm (MOEPGA) which integrates multiple network topological features to detect biologically meaningful protein complexes. Our approach first systematically analyzes the multiobjective problem in terms of identifying protein complexes from PPI networks, and then constructs the objective function of the iterative algorithm based on three common topological properties of protein complexes from the benchmark dataset, finally we describe our algorithm, which mainly consists of three steps, population initialization, subgraph mutation and subgraph selection operation. To show the utility of our method, we compared MOEPGA with several state-of-the-art algorithms on two yeast PPI datasets. The experiment results demonstrate that the proposed method can not only find more protein complexes but also achieve higher accuracy in terms of fscore. Moreover, our approach can cover a certain number of proteins in the input PPI network in terms of the normalized clustering score. Taken together, our method can serve as a powerful framework to detect protein complexes in yeast PPI networks, thereby facilitating the identification of the underlying biological functions.

  5. Mitochondrial Complex I Deficiency Increases Protein Acetylation and Accelerates Heart Failure

    PubMed Central

    Karamanlidis, Georgios; Lee, Chi Fung; Garcia-Menendez, Lorena; Kolwicz, Stephen C.; Suthammarak, Wichit; Gong, Guohua; Sedensky, Margaret M.; Morgan, Philip G.; Wang, Wang; Tian, Rong

    2013-01-01

    Summary Mitochondrial respiratory dysfunction is linked to the pathogenesis of multiple diseases including heart failure but the specific mechanisms for this link remain largely elusive. We modeled the impairment of mitochondrial respiration by inactivation of the Ndufs4 gene, a protein critical for Complex I (C-I) assembly, in the mouse heart (cKO). While C-I supported respiration decreased by >40%, the cKO mice maintained normal cardiac function in vivo and high-energy phosphate content in isolated perfused hearts. However, the cKO mice developed accelerated heart failure after pressure overload or repeated pregnancy. Decreased NAD+/NADH ratio by C-I deficiency inhibited Sirt3 activity, leading to increase in protein acetylation, and sensitization of the permeability transition in mitochondria (mPTP). NAD+ precursor supplementation to cKO mice partially normalized the NAD+/NADH ratio, protein acetylation and mPTP sensitivity. These findings describe a mechanism connecting mitochondrial dysfunction to the susceptibility to diseases and propose a potential therapeutic target. PMID:23931755

  6. The Chaperone Network Connected to Human Ribosome-Associated Complex ▿ ‡ †

    PubMed Central

    Jaiswal, Himjyot; Conz, Charlotte; Otto, Hendrik; Wölfle, Tina; Fitzke, Edith; Mayer, Matthias P.; Rospert, Sabine

    2011-01-01

    Mammalian ribosome-associated complex (mRAC), consisting of the J-domain protein MPP11 and the atypical Hsp70 homolog (70-homolog) Hsp70L1, can partly complement the function of RAC, which is the homologous complex from Saccharomyces cerevisiae. RAC is the J-domain partner exclusively of the 70-homolog Ssb, which directly and independently of RAC binds to the ribosome. We here show that growth defects due to mRAC depletion in HeLa cells resemble those of yeast strains lacking RAC. Functional conservation, however, did not extend to the 70-homolog partner of mRAC. None of the major human 70-homologs was able to complement the growth defects of yeast strains lacking Ssb or was bound to ribosomes in an Ssb-like manner. Instead, our data suggest that mRAC was a specific partner of human Hsp70 but not of its close homolog Hsc70. On a mechanistic level, ATP binding, but not ATP hydrolysis, by Hsp70L1 affected mRAC's function as a J-domain partner of Hsp70. The combined data indicate that, while functionally conserved, yeast and mammalian cells have evolved distinct solutions to ensure that Hsp70-type chaperones can efficiently assist the biogenesis of newly synthesized polypeptide chains. PMID:21245388

  7. Distribution of CPP-Protein Complexes in Freshly Resected Human Tissue Material

    PubMed Central

    Saar, Külliki; Saar, Helgi; Hansen, Mats; Langel, Ülo; Pooga, Margus

    2010-01-01

    Interest in cell-penetrating peptides (CPPs) as delivery agents has fuelled a large number of studies conducted on cultured cells and in mice. However, only a few studies have been devoted to the behaviour of CPPs in human tissues. Therefore, we performed ex vivo tissue-dipping experiments where we studied the distribution of CPP-protein complexes in samples of freshly harvested human tissue material. We used the carcinoma or hyperplasia-containing specimens of the uterus and the cervix, obtained as surgical waste from nine hysterectomies. Our aim was to evaluate the tissue of preference (epithelial versus muscular/connective tissue, carcinoma versus adjacent histologically normal tissue) for two well-studied CPPs, the transportan and the TAT-peptide. We complexed biotinylated CPPs with avidin-β-galactosidase (ABG), which enabled us to apply whole-mount X-gal staining as a robust detection method. Our results demonstrate that both peptides enhanced the tissue distribution of ABG. The enhancing effect of the tested CPPs was more obvious in the normal tissue and in some specimens we detected a striking selectivity of CPP-ABG complexes for the normal tissue. This unexpected finding encourages the evaluation of CPPs as local delivery agents in non-malignant situations, for example in the intrauterine gene therapy of benign gynaecological diseases. PMID:27713271

  8. Chemical cross-linking/mass spectrometry targeting acidic residues in proteins and protein complexes.

    PubMed

    Leitner, Alexander; Joachimiak, Lukasz A; Unverdorben, Pia; Walzthoeni, Thomas; Frydman, Judith; Förster, Friedrich; Aebersold, Ruedi

    2014-07-01

    The study of proteins and protein complexes using chemical cross-linking followed by the MS identification of the cross-linked peptides has found increasingly widespread use in recent years. Thus far, such analyses have used almost exclusively homobifunctional, amine-reactive cross-linking reagents. Here we report the development and application of an orthogonal cross-linking chemistry specific for carboxyl groups. Chemical cross-linking of acidic residues is achieved using homobifunctional dihydrazides as cross-linking reagents and a coupling chemistry at neutral pH that is compatible with the structural integrity of most protein complexes. In addition to cross-links formed through insertion of the dihydrazides with different spacer lengths, zero-length cross-link products are also obtained, thereby providing additional structural information. We demonstrate the application of the reaction and the MS identification of the resulting cross-linked peptides for the chaperonin TRiC/CCT and the 26S proteasome. The results indicate that the targeting of acidic residues for cross-linking provides distance restraints that are complementary and orthogonal to those obtained from lysine cross-linking, thereby expanding the yield of structural information that can be obtained from cross-linking studies and used in hybrid modeling approaches. PMID:24938783

  9. Chemical cross-linking/mass spectrometry targeting acidic residues in proteins and protein complexes

    PubMed Central

    Leitner, Alexander; Joachimiak, Lukasz A.; Unverdorben, Pia; Walzthoeni, Thomas; Frydman, Judith; Förster, Friedrich; Aebersold, Ruedi

    2014-01-01

    The study of proteins and protein complexes using chemical cross-linking followed by the MS identification of the cross-linked peptides has found increasingly widespread use in recent years. Thus far, such analyses have used almost exclusively homobifunctional, amine-reactive cross-linking reagents. Here we report the development and application of an orthogonal cross-linking chemistry specific for carboxyl groups. Chemical cross-linking of acidic residues is achieved using homobifunctional dihydrazides as cross-linking reagents and a coupling chemistry at neutral pH that is compatible with the structural integrity of most protein complexes. In addition to cross-links formed through insertion of the dihydrazides with different spacer lengths, zero-length cross-link products are also obtained, thereby providing additional structural information. We demonstrate the application of the reaction and the MS identification of the resulting cross-linked peptides for the chaperonin TRiC/CCT and the 26S proteasome. The results indicate that the targeting of acidic residues for cross-linking provides distance restraints that are complementary and orthogonal to those obtained from lysine cross-linking, thereby expanding the yield of structural information that can be obtained from cross-linking studies and used in hybrid modeling approaches. PMID:24938783

  10. Microglomerular Synaptic Complexes in the Sky-Compass Network of the Honeybee Connect Parallel Pathways from the Anterior Optic Tubercle to the Central Complex

    PubMed Central

    Held, Martina; Berz, Annuska; Hensgen, Ronja; Muenz, Thomas S.; Scholl, Christina; Rössler, Wolfgang; Homberg, Uwe; Pfeiffer, Keram

    2016-01-01

    While the ability of honeybees to navigate relying on sky-compass information has been investigated in a large number of behavioral studies, the underlying neuronal system has so far received less attention. The sky-compass pathway has recently been described from its input region, the dorsal rim area (DRA) of the compound eye, to the anterior optic tubercle (AOTU). The aim of this study is to reveal the connection from the AOTU to the central complex (CX). For this purpose, we investigated the anatomy of large microglomerular synaptic complexes in the medial and lateral bulbs (MBUs/LBUs) of the lateral complex (LX). The synaptic complexes are formed by tubercle-lateral accessory lobe neuron 1 (TuLAL1) neurons of the AOTU and GABAergic tangential neurons of the central body’s (CB) lower division (TL neurons). Both TuLAL1 and TL neurons strongly resemble neurons forming these complexes in other insect species. We further investigated the ultrastructure of these synaptic complexes using transmission electron microscopy. We found that single large presynaptic terminals of TuLAL1 neurons enclose many small profiles (SPs) of TL neurons. The synaptic connections between these neurons are established by two types of synapses: divergent dyads and divergent tetrads. Our data support the assumption that these complexes are a highly conserved feature in the insect brain and play an important role in reliable signal transmission within the sky-compass pathway. PMID:27774056

  11. Integrating Scientific Content with Context to Connect Educators with the Complexities and Consequences of Climate Change

    NASA Astrophysics Data System (ADS)

    Low, R.; Gosselin, D. C.; Oglesby, R. J.; Larson-Miller, C.; Thomas, J.; Mawalagedara, R.

    2011-12-01

    Over the past three years the Nebraska Earth Systems Education Network has designed professional development opportunities for K-12 and extension educators that integrates scientific content into the context of helping educators connect society with the complexities and consequences of climate change. Our professional development approach uses learner-, knowledge-, assessment-, and community-centered strategies to achieve our long-term goal: collaboration of scientists, educators and learners to foster civic literacy about climate change. Two NASA-funded projects, Global Climate Change Literacy for Educators (GCCE, 2009-2012), and the Educators Climatologists Learning Community (ECLC, 2011-2013), have provided the mechanism to provide teachers with scientifically sound and pedagogically relevant educational materials to improve climate and Earth systems literacy among educators. The primary product of the GCCE program is a 16-week, online, distance-delivered, asynchronous course entitled, Laboratory Earth: Human Dimensions of Climate Change. This course consists of four, four-week modules that integrate climate literacy, Earth Systems concepts, and pedagogy focused on active learning processes, building community, action research, and students' sense of place to promote action at the local level to address the challenges of climate change. Overall, the Community of Inquiry Survey (COI) indicated the course was effective in teaching content, developing a community of learners, and engaging students in experiences designed to develop content knowledge. A pre- and post- course Wilcoxan Signed Ranks Test indicated there was a statistically significant increase in participant's beliefs about their personal science teaching efficacy. Qualitative data from concept maps and content mastery assignments support a positive impact on teachers' content knowledge and classroom practice. Service Learning units seemed tohelp teachers connect course learning to their classroom

  12. EXPERIMENT-GUIDED MOLECULAR MODELING OF PROTEIN-PROTEIN COMPLEXES INVOLVING GPCRS

    PubMed Central

    Kufareva, Irina; Handel, Tracy M.

    2015-01-01

    Summary Experimental structure determination for G protein coupled receptors (GPCRs) and especially their complexes with protein and peptide ligands is at its infancy. In the absence of complex structures, molecular modeling and docking play a large role not only by providing a proper 3D context for interpretation of biochemical and biophysical data, but also by prospectively guiding experiments. Experimentally confirmed restraints may help improve the accuracy and information content of the computational models. Here we present a hybrid molecular modeling protocol that integrates heterogeneous experimental data with force field-based calculations in the stochastic global optimization of the conformations and relative orientations of binding partners. Some experimental data, such as pharmacophore-like chemical fields or disulfide-trapping restraints, can be seamlessly incorporated in the protocol, while other types of data are more useful at the stage of solution filtering. The protocol was successfully applied to modeling and design of a stable construct that resulted in crystallization of the first complex between a chemokine and its receptor. Examples from this work are used to illustrate the steps of the protocol. The utility of different types of experimental data for modeling and docking is discussed and caveats associated with data misinterpretation are highlighted. PMID:26260608

  13. Identifying functions of protein complexes based on topology similarity with random forest.

    PubMed

    Li, Zhan-Chao; Lai, Yan-Hua; Chen, Li-Li; Xie, Yun; Dai, Zong; Zou, Xiao-Yong

    2014-03-01

    Elucidating the functions of protein complexes is critical for understanding disease mechanisms, diagnosis and therapy. In this study, based on the concept that protein complexes with similar topology may have similar functions, we firstly model protein complexes as weighted graphs with nodes representing the proteins and edges indicating interaction between proteins. Secondly, we use topology features derived from the graphs to characterize protein complexes based on the graph theory. Finally, we construct a predictor by using random forest and topology features to identify the functions of protein complexes. Effectiveness of the current method is evaluated by identifying the functions of mammalian protein complexes. And then the predictor is also utilized to identify the functions of protein complexes retrieved from human protein-protein interaction networks. We identify some protein complexes with significant roles in the occurrence of tumors, vesicles and retinoblastoma. It is anticipated that the current research has an important impact on pathogenesis and the pharmaceutical industry. The source code of Matlab and the dataset are freely available on request from the authors. PMID:24389559

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

    PubMed

    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.

  15. A Drosophila ESC-E(Z) protein complex is distinct from other polycomb group complexes and contains covalently modified ESC.

    PubMed

    Ng, J; Hart, C M; Morgan, K; Simon, J A

    2000-05-01

    The extra sex combs (ESC) and Enhancer of zeste [E(Z)] proteins, members of the Polycomb group (PcG) of transcriptional repressors, interact directly and are coassociated in fly embryos. We report that these two proteins are components of a 600-kDa complex in embryos. Using gel filtration and affinity chromatography, we show that this complex is biochemically distinct from previously described complexes containing the PcG proteins Polyhomeotic, Polycomb, and Sex comb on midleg. In addition, we present evidence that ESC is phosphorylated in vivo and that this modified ESC is preferentially associated in the complex with E(Z). Modified ESC accumulates between 2 and 6 h of embryogenesis, which is the developmental time when esc function is first required. We find that mutations in E(z) reduce the ratio of modified to unmodified ESC in vivo. We have also generated germ line transformants that express ESC proteins bearing site-directed mutations that disrupt ESC-E(Z) binding in vitro. These mutant ESC proteins fail to provide esc function, show reduced levels of modification in vivo, and are still assembled into complexes. Taken together, these results suggest that ESC phosphorylation normally occurs after assembly into ESC-E(Z) complexes and that it contributes to the function or regulation of these complexes. We discuss how biochemically separable ESC-E(Z) and PC-PH complexes might work together to provide PcG repression. PMID:10757791

  16. Connectivity among sinkholes and complex networks: The case of Ring of Cenotes in northwest Yucatan, Mexico

    NASA Astrophysics Data System (ADS)

    Gomez-Nicolas, Mariana; Rebolledo-Vieyra, Mario; Huerta-Quintanilla, Rodrigo; Canto-Lugo, Efrain

    2014-05-01

    A 180-km-diameter semicircular alignment of abundant karst sinkholes (locally known as cenotes) in northwestern Yucatán, México, coincides approximately with a concentric ring of the buried Chicxulub structure, a circular feature manifested in Cretaceous and older rocks, that has been identified as the product of the impact of a meteorite. The secondary permeability generated by the fracturing and faulting of the sedimentary sequence in the Chicxulub impact, has favored the karstification process and hence the development of genuine underground rivers that carry water from the continent to the sea. The study of the structure and morphology of the crater has allowed researchers to understand the key role of the crater in the Yucatán hydrogeology. It is generally accepted that the Ring of Cenotes, produced by the gravitational deformation of the Tertiary sedimentary sequence within the crater, controls the groundwater in northern Yucatán. However, today there is not solid evidence about the connectivity among cenotes, which is important because if established, public policies could be designed to manage sanitary infrastructure, septic control, regulation of agricultural and industrial activities and the protection of water that has not been compromised by anthropogenic pollution. All these directly affect more than half a million people whose main source of drinking water lies in the aquifer. In this contribution we investigated a set of 16 cenotes located in the vicinity of a gravimetric anomaly of Chicxulub crater ring, using complex networks to model the interconnectivity among them. Data from a geoelectrical tomography survey, collected with SuperSting R1/IP equipment, with multi-electrodes (72 electrodes), in a dipole-dipole configuration was used as input of our model. Since the total number of cenotes on the ring structure amounts to about 2000, the application of graph theoretic algorithms and Monte Carlo simulation to efficiently investigate network

  17. Analysis of the NuRD subunits reveals a histone deacetylase core complex and a connection with DNA methylation

    PubMed Central

    Zhang, Yi; Ng, Huck-Hui; Erdjument-Bromage, Hediye; Tempst, Paul; Bird, Adrian; Reinberg, Danny

    1999-01-01

    ATP-dependent nucleosome remodeling and core histone acetylation and deacetylation represent mechanisms to alter nucleosome structure. NuRD is a multisubunit complex containing nucleosome remodeling and histone deacetylase activities. The histone deacetylases HDAC1 and HDAC2 and the histone binding proteins RbAp48 and RbAp46 form a core complex shared between NuRD and Sin3-histone deacetylase complexes. The histone deacetylase activity of the core complex is severely compromised. A novel polypeptide highly related to the metastasis-associated protein 1, MTA2, and the methyl-CpG-binding domain-containing protein, MBD3, were found to be subunits of the NuRD complex. MTA2 modulates the enzymatic activity of the histone deacetylase core complex. MBD3 mediates the association of MTA2 with the core histone deacetylase complex. MBD3 does not directly bind methylated DNA but is highly related to MBD2, a polypeptide that binds to methylated DNA and has been reported to possess demethylase activity. MBD2 interacts with the NuRD complex and directs the complex to methylated DNA. NuRD may provide a means of gene silencing by DNA methylation. PMID:10444591

  18. Students' Learning with the Connected Chemistry (CC1) Curriculum: Navigating the Complexities of the Particulate World

    ERIC Educational Resources Information Center

    Levy, Sharona T.; Wilensky, Uri

    2009-01-01

    The focus of this study is students' learning with a Connected Chemistry unit, CC1 (denotes Connected Chemistry, chapter 1), a computer-based environment for learning the topics of gas laws and kinetic molecular theory in chemistry (Levy and Wilensky 2009). An investigation was conducted into high-school students' learning with Connected…

  19. Folding analysis of the most complex Stevedore's protein knot.

    PubMed

    Wang, Iren; Chen, Szu-Yu; Hsu, Shang-Te Danny

    2016-01-01

    DehI is a homodimeric haloacid dehalogenase from Pseudomonas putida that contains the most complex 61 Stevedore's protein knot within its folding topology. To examine how DehI attains such an intricate knotted topology we combined far-UV circular dichroism (CD), intrinsic fluorescence spectroscopy and small angle X-ray scattering (SAXS) to investigate its folding mechanism. Equilibrium unfolding of DehI by chemical denaturation indicated the presence of two highly populated folding intermediates, I and I'. While the two intermediates vary in secondary structure contents and tertiary packing according to CD and intrinsic fluorescence, respectively, their overall dimension and compactness are similar according to SAXS. Three single-tryptophan variants (W34, W53, and W196) were generated to probe non-cooperative unfolding events localized around the three fluorophores. Kinetic fluorescence measurements indicated that the transition from the intermediate I' to the unfolded state is rate limiting. Our multiparametric folding analyses suggest that DehI unfolds through a linear folding pathway with two distinct folding intermediates by initial hydrophobic collapse followed by nucleation condensation, and that knotting precedes the formation of secondary structures. PMID:27527519

  20. Evidence for a vasopressin receptor-GTP binding protein complex

    SciTech Connect

    Fitzgerald, T.J.; Uhing, R.J.; Exton, J.H.

    1986-05-01

    Plasma membranes from the livers of rats were able to hydrolyze the ..gamma..-phosphate from guanosine-5'-triphosphate (GTP). The rate of GTP hydrolysis could be decreased to 10% of its initial rate by the addition of adenosine-5'-triphosphate with a concomitant decrease in the K/sub m/ for GTP from approx. 10/sup -3/ M to 10/sup -6/ M. The low K/sub m/ GTPase activity was inhibited by the addition of nonhydrolyzable analogs of GTP. In addition, the GTPase activity was stimulated from 10 to 30% over basal by the addition of vasopressin. A dose dependency curve showed that the maximum stimulation was obtained with 10/sup -8/ M vasopressin. Identical results were obtained from plasma membranes that had been solubilized with 1% digitonin. When membranes that had been solubilized in the presence of (Phenylalanyl-3,4,5-/sup 3/H(N))vasopressin were subjected to sucrose gradient centrifugation, the majority of bound (/sup 3/H)vasopressin migrated with an approximate molecular weight of 300,000. Moreover, there was a GTPase activity that migrated with the bound (/sup 3/H)vasopressin. This peak of bound (/sup 3/H)vasopressin was decreased by 90% when the sucrose gradient centrifugation was run in the presence of 10/sup -5/ M guanosine-5'-O-(3-thiotriphosphate). These results support the conclusion that liver plasma membranes contain a GTP-binding protein that can complex with the vasopressin receptor.

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  2. The electrodeposition of zinc oxide two-dimensional nanomesh and three-dimensional inverse opal complex connected structures.

    PubMed

    Fu, Ming; Zhou, Ji; Huang, Xueguang; He, Dawei; Wang, Yongsheng

    2010-03-01

    The fabrication of the periodic ordered structures with complex shapes or heterostructures are important for many potential applications. In this paper, zinc oxide two-dimensional (2D) nanomesh and three-dimensional (3D) inverse opal complex connected structures were fabricated by the electrochemical deposition method. 2D ZnO nanosheets or sheet-like porous structures standing on the 3D ZnO inverse opals were synthesized. Two different methods: the co-deposition method and the two-step deposition method were employed for the preparation of the complex structures. The deposition process of complex structures is discussed. The photonic band gap of the complex macroporous structure was calculated using the Translight software and characterized by the micro-region reflection and transmission spectra. The exciton luminescence of ZnO can be inhibited by the inverse opal structure when the photonic band gap superposes with the luminescent band.

  3. Dscam and Sidekick proteins direct lamina-specific synaptic connections in vertebrate retina.

    PubMed

    Yamagata, Masahito; Sanes, Joshua R

    2008-01-24

    Synaptic circuits in the retina transform visual input gathered by photoreceptors into messages that retinal ganglion cells (RGCs) send to the brain. Processes of retinal interneurons (amacrine and bipolar cells) form synapses on dendrites of RGCs in the inner plexiform layer (IPL). The IPL is divided into at least 10 parallel sublaminae; subsets of interneurons and RGCs arborize and form synapses in just one or a few of them. These lamina-specific circuits determine the visual features to which RGC subtypes respond. Here we show that four closely related immunoglobulin superfamily (IgSF) adhesion molecules--Dscam (Down's syndrome cell adhesion molecule), DscamL (refs 6-9), Sidekick-1 and Sidekick-2 (ref. 10)--are expressed in chick by non-overlapping subsets of interneurons and RGCs that form synapses in distinct IPL sublaminae. Moreover, each protein is concentrated within the appropriate sublaminae and each mediates homophilic adhesion. Loss- and gain-of-function studies in vivo indicate that these IgSF members participate in determining the IPL sublaminae in which synaptic partners arborize and connect. Thus, vertebrate Dscams, like Drosophila Dscams, play roles in neural connectivity. Together, our results on Dscams and Sidekicks suggest the existence of an IgSF code for laminar specificity in retina and, by implication, in other parts of the central nervous system. PMID:18216854

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

    PubMed

    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.

  5. 193 nm Ultraviolet Photodissociation Mass Spectrometry of Tetrameric Protein Complexes Provides Insight into Quaternary and Secondary Protein Topology.

    PubMed

    Morrison, Lindsay J; Brodbelt, Jennifer S

    2016-08-31

    Protein-protein interfaces and architecture are critical to the function of multiprotein complexes. Mass spectrometry-based techniques have emerged as powerful strategies for characterization of protein complexes, particularly for heterogeneous mixtures of structures. In the present study, activation and dissociation of three tetrameric protein complexes (streptavidin, transthyretin, and hemoglobin) in the gas phase was undertaken by 193 nm ultraviolet photodissociation (UVPD) for the characterization of higher order structure. High pulse energy UVPD resulted in the production of dimers and low charged monomers exhibiting symmetrical charge partitioning among the subunits (the so-called symmetrical dissociation pathways), consistent with the subunit organization of the complexes. In addition, UVPD promoted backbone cleavages of the monomeric subunits, the abundances of which corresponded to the more flexible loop regions of the proteins. PMID:27480400

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

    PubMed

    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. PMID:22961452

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

    PubMed

    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.

  8. High-throughput Isolation and Characterization of Untagged Membrane Protein Complexes: Outer Membrane Complexes of Desulfovibrio vulgaris

    PubMed Central

    2012-01-01

    Cell membranes represent the “front line” of cellular defense and the interface between a cell and its environment. To determine the range of proteins and protein complexes that are present in the cell membranes of a target organism, we have utilized a “tagless” process for the system-wide isolation and identification of native membrane protein complexes. As an initial subject for study, we have chosen the Gram-negative sulfate-reducing bacterium Desulfovibrio vulgaris. With this tagless methodology, we have identified about two-thirds of the outer membrane- associated proteins anticipated. Approximately three-fourths of these appear to form homomeric complexes. Statistical and machine-learning methods used to analyze data compiled over multiple experiments revealed networks of additional protein–protein interactions providing insight into heteromeric contacts made between proteins across this region of the cell. Taken together, these results establish a D. vulgaris outer membrane protein data set that will be essential for the detection and characterization of environment-driven changes in the outer membrane proteome and in the modeling of stress response pathways. The workflow utilized here should be effective for the global characterization of membrane protein complexes in a wide range of organisms. PMID:23098413

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

    PubMed Central

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

    2015-01-01

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

  10. Tandem Affinity Purification of Protein Complexes in Mouse Embryonic Stem Cells Using In Vivo Biotinylation

    PubMed Central

    Wang, Jianlong; Cantor, Alan B.; Orkin, Stuart H.

    2009-01-01

    In dissecting the pluripotent state in mouse embryonic stem (ES) cells, we have employed in vivo biotinylation of critical transcription factors for streptavidin affinity purification of protein complexes and constructed a protein-protein interaction network. This has facilitated discovery of novel pluripotency factors and a better understanding of stem cell pluripotency. Here we describe detailed procedures for in vivo biotinylation system setup in mouse ES cells and affinity purification of multi-protein complexes using in vivo biotinylation. In addition, we present a protocol employing SDS-PAGE fractionation to reduce sample complexity prior to submission for mass spectrometry (MS) protein identification. PMID:19306258

  11. Protein tyrosine phosphatase σ targets apical junction complex proteins in the intestine and regulates epithelial permeability.

    PubMed

    Murchie, Ryan; Guo, Cong-Hui; Persaud, Avinash; Muise, Aleixo; Rotin, Daniela

    2014-01-14

    Protein tyrosine phosphatase (PTP)σ (PTPRS) was shown previously to be associated with susceptibility to inflammatory bowel disease (IBD). PTPσ(-/-) mice exhibit an IBD-like phenotype in the intestine and show increased susceptibility to acute models of murine colitis. However, the function of PTPσ in the intestine is uncharacterized. Here, we show an intestinal epithelial barrier defect in the PTPσ(-/-) mouse, demonstrated by a decrease in transepithelial resistance and a leaky intestinal epithelium that was determined by in vivo tracer analysis. Increased tyrosine phosphorylation was observed at the plasma membrane of epithelial cells lining the crypts of the small bowel and colon of the PTPσ(-/-) mouse, suggesting the presence of PTPσ substrates in these regions. Using mass spectrometry, we identified several putative PTPσ intestinal substrates that were hyper-tyrosine-phosphorylated in the PTPσ(-/-) mice relative to wild type. Among these were proteins that form or regulate the apical junction complex, including ezrin. We show that ezrin binds to and is dephosphorylated by PTPσ in vitro, suggesting it is a direct PTPσ substrate, and identified ezrin-Y353/Y145 as important sites targeted by PTPσ. Moreover, subcellular localization of the ezrin phosphomimetic Y353E or Y145 mutants were disrupted in colonic Caco-2 cells, similar to ezrin mislocalization in the colon of PTPσ(-/-) mice following induction of colitis. Our results suggest that PTPσ is a positive regulator of intestinal epithelial barrier, which mediates its effects by modulating epithelial cell adhesion through targeting of apical junction complex-associated proteins (including ezrin), a process impaired in IBD.

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

    PubMed

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

    2013-05-01

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

  13. ConPlex: a server for the evolutionary conservation analysis of protein complex structures.

    PubMed

    Choi, Yoon Sup; Han, Seong Kyu; Kim, Jinho; Yang, Jae-Seong; Jeon, Jouhyun; Ryu, Sung Ho; Kim, Sanguk

    2010-07-01

    Evolutionary conservation analyses are important for the identification of protein-protein interactions. For protein complex structures, sequence conservation has been applied to determine protein oligomerization states, to characterize native interfaces from non-specific crystal contacts, and to discriminate near-native structures from docking artifacts. However, a user-friendly web-based service for evolutionary conservation analysis of protein complexes has not been available. Therefore, we developed ConPlex (http://sbi.postech.ac.kr/ConPlex/) a web application that enables evolutionary conservation analyses of protein interactions within protein quaternary structures. Users provide protein complex structures; ConPlex automatically identifies protein interfaces and carries out evolutionary conservation analyses for the interface regions. Moreover, ConPlex allows the results of the residue-specific conservation analysis to be displayed on the protein complex structure and provides several options to customize the display output to fit each user's needs. We believe that ConPlex offers a convenient platform to analyze protein complex structures based on evolutionary conservation of protein-protein interface residues.

  14. Enhanced conformational sampling to visualize a free-energy landscape of protein complex formation

    PubMed Central

    Iida, Shinji; Nakamura, Haruki; Higo, Junichi

    2016-01-01

    We introduce various, recently developed, generalized ensemble methods, which are useful to sample various molecular configurations emerging in the process of protein–protein or protein–ligand binding. The methods introduced here are those that have been or will be applied to biomolecular binding, where the biomolecules are treated as flexible molecules expressed by an all-atom model in an explicit solvent. Sampling produces an ensemble of conformations (snapshots) that are thermodynamically probable at room temperature. Then, projection of those conformations to an abstract low-dimensional space generates a free-energy landscape. As an example, we show a landscape of homo-dimer formation of an endothelin-1-like molecule computed using a generalized ensemble method. The lowest free-energy cluster at room temperature coincided precisely with the experimentally determined complex structure. Two minor clusters were also found in the landscape, which were largely different from the native complex form. Although those clusters were isolated at room temperature, with rising temperature a pathway emerged linking the lowest and second-lowest free-energy clusters, and a further temperature increment connected all the clusters. This exemplifies that the generalized ensemble method is a powerful tool for computing the free-energy landscape, by which one can discuss the thermodynamic stability of clusters and the temperature dependence of the cluster networks. PMID:27288028

  15. Dissecting the function of Atg1 complex in Dictyostelium autophagy reveals a connection with the pentose phosphate pathway enzyme transketolase.

    PubMed

    Mesquita, Ana; Tábara, Luis C; Martinez-Costa, Oscar; Santos-Rodrigo, Natalia; Vincent, Olivier; Escalante, Ricardo

    2015-08-01

    The network of protein-protein interactions of the Dictyostelium discoideum autophagy pathway was investigated by yeast two-hybrid screening of the conserved autophagic proteins Atg1 and Atg8. These analyses confirmed expected interactions described in other organisms and also identified novel interactors that highlight the complexity of autophagy regulation. The Atg1 kinase complex, an essential regulator of autophagy, was investigated in detail here. The composition of the Atg1 complex in D. discoideum is more similar to mammalian cells than to Saccharomyces cerevisiae as, besides Atg13, it contains Atg101, a protein not conserved in this yeast. We found that Atg101 interacts with Atg13 and genetic disruption of these proteins in Dictyostelium leads to an early block in autophagy, although the severity of the developmental phenotype and the degree of autophagic block is higher in Atg13-deficient cells. We have also identified a protein containing zinc-finger B-box and FNIP motifs that interacts with Atg101. Disruption of this protein increases autophagic flux, suggesting that it functions as a negative regulator of Atg101. We also describe the interaction of Atg1 kinase with the pentose phosphate pathway enzyme transketolase (TKT). We found changes in the activity of endogenous TKT activity in strains lacking or overexpressing Atg1, suggesting the presence of an unsuspected regulatory pathway between autophagy and the pentose phosphate pathway in Dictyostelium that seems to be conserved in mammalian cells.

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

    NASA Astrophysics Data System (ADS)

    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.

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

    SciTech Connect

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

    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.

  18. Detection and identification of protein citrullination in complex biological systems.

    PubMed

    Clancy, Kathleen W; Weerapana, Eranthie; Thompson, Paul R

    2016-02-01

    Protein citrullination is a post-translational modification of arginine that is catalyzed by the Protein Arginine Deiminase (PAD) family of enzymes. Aberrantly increased citrullination is associated with a host of inflammatory diseases and cancer and PAD inhibitors have shown remarkable efficacy in a range of diseases including rheumatoid arthritis, lupus, atherosclerosis, and ulcerative colitis. In rheumatoid arthritis, citrullinated proteins serve as key antigens for rheumatoid arthritis-associated autoantibodies. These data suggest that citrullinated proteins may serve more generally as biomarkers of specific disease states, however, the identification of citrullinated residues remains challenging due to the small 1Da mass change that occurs upon citrullination. Herein, we highlight the available techniques to identify citrullinated proteins/residues focusing on advanced MS techniques as well as chemical derivatization strategies that are currently being employed to identify citrullinated proteins as well as the specific residues modified within those proteins.

  19. C1orf163/RESA1 is a novel mitochondrial intermembrane space protein connected to respiratory chain assembly.

    PubMed

    Kozjak-Pavlovic, Vera; Prell, Florian; Thiede, Bernd; Götz, Monika; Wosiek, Dominik; Ott, Christine; Rudel, Thomas

    2014-02-20

    Oxidative phosphorylation (OXPHOS) in mitochondria takes place at the inner membrane, which folds into numerous cristae. The stability of cristae depends, among other things, on the mitochondrial intermembrane space bridging complex. Its components include inner mitochondrial membrane protein mitofilin and outer membrane protein Sam50. We identified a conserved, uncharacterized protein, C1orf163 [SEL1 repeat containing 1 protein (SELRC1)], as one of the proteins significantly reduced after the knockdown of Sam50 and mitofilin. We show that C1orf163 is a mitochondrial soluble intermembrane space protein. Sam50 depletion affects moderately the import and assembly of C1orf163 into two protein complexes of approximately 60kDa and 150kDa. We observe that the knockdown of C1orf163 leads to reduction of levels of proteins belonging to the OXPHOS complexes. The activity of complexes I and IV is reduced in C1orf163-depleted cells, and we observe the strongest defects in the assembly of complex IV. Therefore, we propose C1orf163 to be a novel factor important for the assembly of respiratory chain complexes in human mitochondria and suggest to name it RESA1 (for RESpiratory chain Assembly 1).

  20. Reinforcing the LINC complex connection to actin filaments: the role of FHOD1 in TAN line formation and nuclear movement

    PubMed Central

    Antoku, Susumu; Zhu, Ruijun; Kutscheidt, Stefan; Fackler, Oliver T; Gundersen, Gregg G

    2015-01-01

    Positioning the nucleus is critical for many cellular processes including cell division, migration and differentiation. The linker of nucleoskeleton and cytoskeleton (LINC) complex spans the inner and outer nuclear membranes and has emerged as a major factor in connecting the nucleus to the cytoskeleton for movement and positioning. Recently, we discovered that the diaphanous formin family member FHOD1 interacts with the LINC complex component nesprin-2 giant (nesprin-2G) and that this interaction plays essential roles in the formation of transmembrane actin-dependent nuclear (TAN) lines and nuclear movement during cell polarization in fibroblasts. We found that FHOD1 strengthens the connection between nesprin-2G and rearward moving dorsal actin cables by providing a second site of interaction between nesprin-2G and the actin cable. These results indicate that the LINC complex connection to the actin cytoskeleton can be enhanced by cytoplasmic factors and suggest a new model for TAN line formation. We discuss how the nesprin-2G-FHOD1 interaction may be regulated and its possible functional significance for development and disease. PMID:26083340

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

    SciTech Connect

    Bailey, Lucas J.; Fox, Brian G.

    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.

  2. Complex interaction of dendritic connectivity and hierarchical patch size on biodiversity in river-like landscapes.

    PubMed

    Carrara, Francesco; Rinaldo, Andrea; Giometto, Andrea; Altermatt, Florian

    2014-01-01

    Habitat fragmentation and land use changes are causing major biodiversity losses. Connectivity of the landscape or environmental conditions alone can shape biodiversity patterns. In nature, however, local habitat characteristics are often intrinsically linked to a specific connectivity. Such a link is evident in riverine ecosystems, where hierarchical dendritic structures command related scaling on habitat capacity. We experimentally disentangled the effect of local habitat capacity (i.e., the patch size) and dendritic connectivity on biodiversity in aquatic microcosm metacommunities by suitably arranging patch sizes within river-like networks. Overall, more connected communities that occupy a central position in the network exhibited higher species richness, irrespective of patch size arrangement. High regional evenness in community composition was found only in landscapes preserving geomorphological scaling properties of patch sizes. In these landscapes, some of the rarer species sustained regionally more abundant populations better tracking their own niche requirements compared to landscapes with homogeneous patch size or landscapes with spatially uncorrelated patch size. Our analysis suggests that altering the natural link between dendritic connectivity and patch size strongly affects community composition and population persistence at multiple scales. The experimental results are demonstrating a principle that can be tested in theoretical metacommunity models and eventually be projected to real riverine ecosystems.

  3. Tuning structure of oppositely charged nanoparticle and protein complexes

    SciTech Connect

    Kumar, Sugam Aswal, V. K.; Callow, P.

    2014-04-24

    Small-angle neutron scattering (SANS) has been used to probe the structures of anionic silica nanoparticles (LS30) and cationic lyszyme protein (M.W. 14.7kD, I.P. ∼ 11.4) by tuning their interaction through the pH variation. The protein adsorption on nanoparticles is found to be increasing with pH and determined by the electrostatic attraction between two components as well as repulsion between protein molecules. We show the strong electrostatic attraction between nanoparticles and protein molecules leads to protein-mediated aggregation of nanoparticles which are characterized by fractal structures. At pH 5, the protein adsorption gives rise to nanoparticle aggregation having surface fractal morphology with close packing of nanoparticles. The surface fractals transform to open structures of mass fractal morphology at higher pH (7 and 9) on approaching isoelectric point (I.P.)

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

    PubMed

    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. A 1 MDa protein complex containing critical components of the Escherichia coli divisome

    PubMed Central

    Trip, Erik N.; Scheffers, Dirk-Jan

    2015-01-01

    Cell division in bacteria is an essential process that is carried out at mid-cell by a group of cell division proteins referred to as the divisome. In Escherichia coli, over two dozen cell division proteins have been identified of which ten are essential. These division proteins localize sequentially and interdependently to the division site, after which constriction eventually produces two daughter cells. Various genetic and biochemical techniques have identified many interactions amongst cell division proteins, however the existence of the divisome as a large multi-protein complex has never been shown. Here, we identify a 1 MDa protein complex by native page that contains seven essential cell division proteins (FtsZ, ZipA, FtsK, FtsQ, FtsB, FtsL, and FtsN). The 1 MDa complex is present in rapidly dividing cells, but absent when cultures enter the stationary growth phase. Slight overexpression of the ftsQ D237N mutation that blocks cell division prevents formation of this 1 MDa complex. In cells depleted of FtsN, the 1 MDa complex is not assembled. Combined, our findings indicate that a large protein complex containing many different cell division proteins indeed exists. We note that this complex is very fragile and sensitive to the expression of tagged versions of FtsQ. PMID:26643979

  6. A Proteomic Strategy for Global Analysis of Plant Protein Complexes[W][OPEN

    PubMed Central

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

    2014-01-01

    Global analyses of protein complex assembly, composition, and location are needed to fully understand how cells coordinate diverse metabolic, mechanical, and developmental activities. The most common methods for proteome-wide analysis of protein complexes rely on affinity purification-mass spectrometry or yeast two-hybrid approaches. These methods are time consuming and are not suitable for many plant species that are refractory to transformation or genome-wide cloning of open reading frames. Here, we describe the proof of concept for a method allowing simultaneous global analysis of endogenous protein complexes that begins with intact leaves and combines chromatographic separation of extracts from subcellular fractions with quantitative label-free protein abundance profiling by liquid chromatography-coupled mass spectrometry. Applying this approach to the crude cytosolic fraction of Arabidopsis thaliana leaves using size exclusion chromatography, we identified hundreds of cytosolic proteins that appeared to exist as components of stable protein complexes. The reliability of the method was validated by protein immunoblot analysis and comparisons with published size exclusion chromatography data and the masses of known complexes. The method can be implemented with appropriate instrumentation, is applicable to any biological system, and has the potential to be further developed to characterize the composition of protein complexes and measure the dynamics of protein complex localization and assembly under different conditions. PMID:25293756

  7. Dissecting the function of Atg1 complex in Dictyostelium autophagy reveals a connection with the pentose phosphate pathway enzyme transketolase

    PubMed Central

    Mesquita, Ana; Tábara, Luis C.; Martinez-Costa, Oscar; Santos-Rodrigo, Natalia; Vincent, Olivier; Escalante, Ricardo

    2015-01-01

    The network of protein–protein interactions of the Dictyostelium discoideum autophagy pathway was investigated by yeast two-hybrid screening of the conserved autophagic proteins Atg1 and Atg8. These analyses confirmed expected interactions described in other organisms and also identified novel interactors that highlight the complexity of autophagy regulation. The Atg1 kinase complex, an essential regulator of autophagy, was investigated in detail here. The composition of the Atg1 complex in D. discoideum is more similar to mammalian cells than to Saccharomyces cerevisiae as, besides Atg13, it contains Atg101, a protein not conserved in this yeast. We found that Atg101 interacts with Atg13 and genetic disruption of these proteins in Dictyostelium leads to an early block in autophagy, although the severity of the developmental phenotype and the degree of autophagic block is higher in Atg13-deficient cells. We have also identified a protein containing zinc-finger B-box and FNIP motifs that interacts with Atg101. Disruption of this protein increases autophagic flux, suggesting that it functions as a negative regulator of Atg101. We also describe the interaction of Atg1 kinase with the pentose phosphate pathway enzyme transketolase (TKT). We found changes in the activity of endogenous TKT activity in strains lacking or overexpressing Atg1, suggesting the presence of an unsuspected regulatory pathway between autophagy and the pentose phosphate pathway in Dictyostelium that seems to be conserved in mammalian cells. PMID:26246495

  8. Theory of polyelectrolyte adsorption on heterogeneously charged surfaces applied to soluble protein-polyelectrolyte complexes

    NASA Astrophysics Data System (ADS)

    de Vries, R.; Weinbreck, F.; de Kruif, C. G.

    2003-03-01

    Existing theoretical approaches to polymer adsorption on heterogeneous surfaces are applied to the problems of polyelectrolyte and polyampholyte adsorption on randomly charged surfaces. Also, analytical estimates are developed for the critical pH at which weakly charged polyelectrolytes and globular proteins start forming soluble complexes. Below a critical salt concentration, soluble complexes form "on the wrong side" of the protein isoelectric point due to the heterogeneity of the protein surface charge distribution. The analytical estimates are consistent with experimental data on soluble complexes in mixtures of gum arabic and whey protein isolate.

  9. Novel type of red-shifted chlorophyll a antenna complex from Chromera velia. I. Physiological relevance and functional connection to photosystems.

    PubMed

    Kotabová, Eva; Jarešová, Jana; Kaňa, Radek; Sobotka, Roman; Bína, David; Prášil, Ondřej

    2014-06-01

    Chromera velia is an alveolate alga associated with scleractinian corals. Here we present detailed work on chromatic adaptation in C. velia cultured under either blue or red light. Growth of C. velia under red light induced the accumulation of a light harvesting antenna complex exhibiting unusual spectroscopic properties with red-shifted absorption and atypical 710nm fluorescence emission at room temperature. Due to these characteristic features the complex was designated "Red-shifted Chromera light harvesting complex" (Red-CLH complex). Its detailed biochemical survey is described in the accompanying paper (Bina et al. 2013, this issue). Here, we show that the accumulation of Red-CLH complex under red light represents a slow acclimation process (days) that is reversible with much faster kinetics (hours) under blue light. This chromatic adaptation allows C. velia to maintain all important parameters of photosynthesis constant under both light colors. We further demonstrated that the C. velia Red-CLH complex is assembled from a 17kDa antenna protein and is functionally connected to photosystem II as it shows variability of chlorophyll fluorescence. Red-CLH also serves as an additional locus for non-photochemical quenching. Although overall rates of oxygen evolution and carbon fixation were similar for both blue and red light conditions, the presence of Red-CLH in C. velia cells increases the light harvesting potential of photosystem II, which manifested as a doubled oxygen evolution rate at illumination above 695nm. This data demonstrates a remarkable long-term remodeling of C. velia light-harvesting system according to light quality and suggests physiological significance of 'red' antenna complexes.

  10. Surfactant-free purification of membrane protein complexes from bacteria: application to the staphylococcal penicillin-binding protein complex PBP2/PBP2a

    NASA Astrophysics Data System (ADS)

    Paulin, Sarah; Jamshad, Mohammed; Dafforn, Timothy R.; Garcia-Lara, Jorge; Foster, Simon J.; Galley, Nicola F.; Roper, David I.; Rosado, Helena; Taylor, Peter W.

    2014-07-01

    Surfactant-mediated removal of proteins from biomembranes invariably results in partial or complete loss of function and disassembly of multi-protein complexes. We determined the capacity of styrene-co-maleic acid (SMA) co-polymer to remove components of the cell division machinery from the membrane of drug-resistant staphylococcal cells. SMA-lipid nanoparticles solubilized FtsZ-PBP2-PBP2a complexes from intact cells, demonstrating the close physical proximity of these proteins within the lipid bilayer. Exposure of bacteria to (-)-epicatechin gallate, a polyphenolic agent that abolishes β-lactam resistance in staphylococci, disrupted the association between PBP2 and PBP2a. Thus, SMA purification provides a means to remove native integral membrane protein assemblages with minimal physical disruption and shows promise as a tool for the interrogation of molecular aspects of bacterial membrane protein structure and function.

  11. Production of unstable proteins through the formation of stable core complexes.

    PubMed

    Levy, Nicolas; Eiler, Sylvia; Pradeau-Aubreton, Karine; Maillot, Benoit; Stricher, François; Ruff, Marc

    2016-01-01

    Purification of proteins that participate in large transient complexes is impeded by low amounts, heterogeneity, instability and poor solubility. To circumvent these difficulties we set up a methodology that enables the production of stable complexes for structural and functional studies. This procedure is benchmarked and applied to two challenging protein families: the human steroid nuclear receptors (SNR) and the HIV-1 pre-integration complex. In the context of transcriptional regulation studies, we produce and characterize the ligand-binding domains of the glucocorticoid nuclear receptor and the oestrogen receptor beta in complex with a TIF2 (transcriptional intermediary factor 2) domain containing the three SNR-binding motifs. In the context of retroviral integration, we demonstrate the stabilization of the HIV-1 integrase by formation of complexes with partner proteins and DNA. This procedure provides a powerful research tool for structural and functional studies of proteins participating in non-covalent macromolecular complexes. PMID:26983699

  12. Production of unstable proteins through the formation of stable core complexes

    PubMed Central

    Levy, Nicolas; Eiler, Sylvia; Pradeau-Aubreton, Karine; Maillot, Benoit; Stricher, François; Ruff, Marc

    2016-01-01

    Purification of proteins that participate in large transient complexes is impeded by low amounts, heterogeneity, instability and poor solubility. To circumvent these difficulties we set up a methodology that enables the production of stable complexes for structural and functional studies. This procedure is benchmarked and applied to two challenging protein families: the human steroid nuclear receptors (SNR) and the HIV-1 pre-integration complex. In the context of transcriptional regulation studies, we produce and characterize the ligand-binding domains of the glucocorticoid nuclear receptor and the oestrogen receptor beta in complex with a TIF2 (transcriptional intermediary factor 2) domain containing the three SNR-binding motifs. In the context of retroviral integration, we demonstrate the stabilization of the HIV-1 integrase by formation of complexes with partner proteins and DNA. This procedure provides a powerful research tool for structural and functional studies of proteins participating in non-covalent macromolecular complexes. PMID:26983699

  13. Structural Basis for the Coevolution of a Viral RNA-Protein Complex

    SciTech Connect

    Chao,J.; Patskovsky, Y.; Almo, S.; Singer, R.

    2008-01-01

    The cocrystal structure of the PP7 bacteriophage coat protein in complex with its translational operator identifies a distinct mode of sequence-specific RNA recognition when compared to the well-characterized MS2 coat protein-RNA complex. The structure reveals the molecular basis of the PP7 coat protein's ability to selectively bind its cognate RNA, and it demonstrates that the conserved beta-sheet surface is a flexible architecture that can evolve to recognize diverse RNA hairpins.

  14. Determination of the structure and morphology of gold nanoparticle-HSA protein complexes.

    PubMed

    Capomaccio, Robin; Jimenez, Isaac Ojea; Colpo, Pascal; Gilliland, Douglas; Ceccone, Giacomo; Rossi, François; Calzolai, Luigi

    2015-11-14

    We propose a simple method to determine the structure and morphology of nanoparticle protein complexes. By combining a separation method with online size measurements, density measurements and circular dichroism, we could identify the number of proteins bound to each nanoparticle and their secondary structure changes in the complex. This method provides much-needed experimental information on the interaction of proteins with nanoparticles and on the behavior of nanoparticles in biological systems. PMID:26462441

  15. Protein Nanocages for Delivery and Release of Luminescent Ruthenium(II) Polypyridyl Complexes.

    PubMed

    Li, Xiao; Zhang, Yajie; Chen, Hong; Sun, Jian; Feng, Fude

    2016-09-01

    In this report, noncovalent encapsulation of hydrophobic ruthenium(II) polyridyl complexes, Ru(bpy)2dppz(2+) and Ru(phen)2dppz(2+), into apoferritin cavity was achieved with high loading contents by effective prevention of Ru complex-induced protein aggregation, without disruption of protein native architecture. The Ru-loaded luminescent nanocomposites have demonstrated improved water solubility, easy manipulation, reduced cytotoxicity, and enhanced cellular uptake as compared to the nontreated Ru complexes. PMID:27547981

  16. Basement membrane and connective tissue proteins in intestinal mucosa of patients with coeliac disease

    PubMed Central

    Verbeke, S; Gotteland, M; Fernández, M; Bremer, J; Ríos, G; Brunser, O

    2002-01-01

    Aims: Gluten ingestion in coeliac disease is associated with alterations of the intestinal mucosa, especially the expansion of the lamina propria. Antiendomysium and antireticulin antibodies may result from interactions between gliadin and extracellular matrix components. By behaving as autoantigens, connective tissue proteins could initiate mucosal damage. This study evaluates changes in the distribution of laminin, type IV collagen, and fibronectin in the mucosa of patients with coeliac disease in an attempt to explain the alterations of mucosal morphology. Methods: Intestinal biopsies were obtained from patients with coeliac disease on admission and while on a gluten free diet. The distribution of type IV collagen, laminin, fibronectin, and α-smooth muscle actin was evaluated by immunofluorescence and by immunogold labelling and electron microscopy. Results: In patients with coeliac disease, the intensity of type IV collagen, laminin, and fibronectin immunofluorescent staining was decreased and less well defined than in controls, with frequent breaches in the basement membrane; fibronectin staining was weak in the distal third of the elongated crypts and absent under the flat surface. The distribution of smooth muscle fibre in the distal lamina propria of flat mucosae was altered. The distribution of these proteins was normal as assessed by immunoelectron microscopy. Conclusions: The intensity of staining of some components of the basement membrane is decreased in coeliac disease and the distribution of smooth muscle fibres is altered. These changes may result from interactions between gliadin and components of the extracellular matrix and may play a role in the genesis of mucosal lesions and in the damage to the epithelium. PMID:12037027

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

    PubMed

    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.

  18. Removal of lipopolysaccharides from protein-lipopolysaccharide complexes by nonflammable solvents.

    PubMed

    Lin, Miao-Fang; Williams, Christie; Murray, Michael V; Ropp, Philip A

    2005-02-25

    During the recovery of recombinant proteins from gram negative bacteria, many of the methods used to extract proteins from cells release lipopolysaccharides (LPS, endotoxin) along with the protein of interest. In many instances, LPS will co-purify with the target protein due to specific or non-specific protein-LPS interactions. We have investigated the ability of alkanediols to effect the separation of LPS from protein-LPS complexes while the complexes are immobilized on ion exchange chromatographic resins. Proteins were complexed with fluorescently labeled LPS and bound to ion exchange resin. Alkanediol washes of the resins were preformed and the proteins eluted. Column eluates were monitored for LPS and protein by fluorescence and UV spectroscopy, respectively. Alkanediols were effective agents for dissociating LPS from protein-LPS complexes. The efficiency of LPS removal increased with increasing alkanediol chain length. The 1,2-alkanediol isomers were more effective than terminal alkanediol isomers in the separation of LPS from protein-LPS complexes, while the separation of LPS from protein-LPS complexes was more efficient on cation exchangers than on anion exchangers. In addition, it was noted during these investigations that the 1,2-alkanediols increased the retention time of the proteins on the ion exchange resins. Alkanediols provide a safer alternative to the use of other organics such as alcohols or acetonitrile for the separation of LPS from protein due to their lower toxicity and decreased inflammability. In addition, they are less costly than many of the detergents that have been used for similar purposes.

  19. GPCR: G protein complexes--the fundamental signaling assembly.

    PubMed

    Jastrzebska, Beata

    2013-12-01

    G protein coupled receptors (GPCR) constitute the largest group of cell surface receptors that transmit various signals across biological membranes through the binding and activation of heterotrimeric G proteins, which amplify the signal and activate downstream effectors leading to the biological responses. Thus, the first critical step in this signaling cascade is the interaction between receptor and its cognate G protein. Understanding this critical event at the molecular level is of high importance because abnormal function of GPCRs is associated with many diseases. Thus, these receptors are targets for drug development.

  20. A single Sec61-complex functions as a protein-conducting channel.

    PubMed

    Kalies, Kai-Uwe; Stokes, Vivica; Hartmann, Enno

    2008-12-01

    During cotranslational translocation of proteins into the endoplasmic reticulum (ER) translating ribosomes bind to Sec61-complexes. Presently two models exist how these membrane protein complexes might form protein-conducting channels. While electron microscopic data suggest that a ring-like structure consisting of four Sec61-complexes build the channel, the recently solved crystal structure of a homologous bacterial protein complex led to the speculation that the actual tunnel is formed by just one individual Sec61-complex. Using protease protection assays together with quantitative immunoblotting we directly examined the structure of mammalian protein-conducting channels. We found that in native ER-membranes one single Sec61alpha-molecule is preferentially protected by a membrane bound ribosome, both, in the presence and absence of nascent polypeptides. In addition we present evidence that the nascent polypeptide destabilizes the ring-like translocation apparatus formed by four Sec61-complexes. Moreover, we found that after solubilization of ER-membranes a single Sec61-complex is sufficient to protect the nascent polypeptide chain against added proteases. Finally, we could show that this single Sec61-complex allows the movement of the nascent chain, when it has been released from the ribosome by puromycin treatment. Collectively, our data suggest that the active protein-conducting channel in the ER is formed by a single Sec61-complex.

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

    PubMed

    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/.

  2. Control of Synaptic Connectivity by a Network of Drosophila IgSF Cell Surface Proteins.

    PubMed

    Carrillo, Robert A; Özkan, Engin; Menon, Kaushiki P; Nagarkar-Jaiswal, Sonal; Lee, Pei-Tseng; Jeon, Mili; Birnbaum, Michael E; Bellen, Hugo J; Garcia, K Christopher; Zinn, Kai

    2015-12-17

    We have defined a network of interacting Drosophila cell surface proteins in which a 21-member IgSF subfamily, the Dprs, binds to a nine-member subfamily, the DIPs. The structural basis of the Dpr-DIP interaction code appears to be dictated by shape complementarity within the Dpr-DIP binding interface. Each of the six dpr and DIP genes examined here is expressed by a unique subset of larval and pupal neurons. In the neuromuscular system, interactions between Dpr11 and DIP-γ affect presynaptic terminal development, trophic factor responses, and neurotransmission. In the visual system, dpr11 is selectively expressed by R7 photoreceptors that use Rh4 opsin (yR7s). Their primary synaptic targets, Dm8 amacrine neurons, express DIP-γ. In dpr11 or DIP-γ mutants, yR7 terminals extend beyond their normal termination zones in layer M6 of the medulla. DIP-γ is also required for Dm8 survival or differentiation. Our findings suggest that Dpr-DIP interactions are important determinants of synaptic connectivity.

  3. Determining Protein Complex Structures Based on a Bayesian Model of in Vivo Förster Resonance Energy Transfer (FRET) Data*

    PubMed Central

    Bonomi, Massimiliano; Pellarin, Riccardo; Kim, Seung Joong; Russel, Daniel; Sundin, Bryan A.; Riffle, Michael; Jaschob, Daniel; Ramsden, Richard; Davis, Trisha N.; Muller, Eric G. D.; Sali, Andrej

    2014-01-01

    The use of in vivo Förster resonance energy transfer (FRET) data to determine the molecular architecture of a protein complex in living cells is challenging due to data sparseness, sample heterogeneity, signal contributions from multiple donors and acceptors, unequal fluorophore brightness, photobleaching, flexibility of the linker connecting the fluorophore to the tagged protein, and spectral cross-talk. We addressed these challenges by using a Bayesian approach that produces the posterior probability of a model, given the input data. The posterior probability is defined as a function of the dependence of our FRET metric FRETR on a structure (forward model), a model of noise in the data, as well as prior information about the structure, relative populations of distinct states in the sample, forward model parameters, and data noise. The forward model was validated against kinetic Monte Carlo simulations and in vivo experimental data collected on nine systems of known structure. In addition, our Bayesian approach was validated by a benchmark of 16 protein complexes of known structure. Given the structures of each subunit of the complexes, models were computed from synthetic FRETR data with a distance root-mean-squared deviation error of 14 to 17 Å. The approach is implemented in the open-source Integrative Modeling Platform, allowing us to determine macromolecular structures through a combination of in vivo FRETR data and data from other sources, such as electron microscopy and chemical cross-linking. PMID:25139910

  4. Determining protein complex structures based on a Bayesian model of in vivo Förster resonance energy transfer (FRET) data.

    PubMed

    Bonomi, Massimiliano; Pellarin, Riccardo; Kim, Seung Joong; Russel, Daniel; Sundin, Bryan A; Riffle, Michael; Jaschob, Daniel; Ramsden, Richard; Davis, Trisha N; Muller, Eric G D; Sali, Andrej

    2014-11-01

    The use of in vivo Förster resonance energy transfer (FRET) data to determine the molecular architecture of a protein complex in living cells is challenging due to data sparseness, sample heterogeneity, signal contributions from multiple donors and acceptors, unequal fluorophore brightness, photobleaching, flexibility of the linker connecting the fluorophore to the tagged protein, and spectral cross-talk. We addressed these challenges by using a Bayesian approach that produces the posterior probability of a model, given the input data. The posterior probability is defined as a function of the dependence of our FRET metric FRETR on a structure (forward model), a model of noise in the data, as well as prior information about the structure, relative populations of distinct states in the sample, forward model parameters, and data noise. The forward model was validated against kinetic Monte Carlo simulations and in vivo experimental data collected on nine systems of known structure. In addition, our Bayesian approach was validated by a benchmark of 16 protein complexes of known structure. Given the structures of each subunit of the complexes, models were computed from synthetic FRETR data with a distance root-mean-squared deviation error of 14 to 17 Å. The approach is implemented in the open-source Integrative Modeling Platform, allowing us to determine macromolecular structures through a combination of in vivo FRETR data and data from other sources, such as electron microscopy and chemical cross-linking.

  5. Protein-protein interactions indicate composition of a 480 kDa SELMA complex in the second outermost membrane of diatom complex plastids.

    PubMed

    Lau, Julia B; Stork, Simone; Moog, Daniel; Schulz, Julian; Maier, Uwe G

    2016-04-01

    Most secondary plastids of red algal origin are surrounded by four membranes and nucleus-encoded plastid proteins have to traverse these barriers. Translocation across the second outermost plastid membrane, the periplastidal membrane (PPM), is facilitated by a ERAD-(ER-associated degradation) derived machinery termed SELMA (symbiont-specific ERAD-like machinery). In the last years, important subunits of this translocator have been identified, which clearly imply compositional similarities between SELMA and ERAD. Here we investigated, via protein-protein interaction studies, if the composition of SELMA is comparable to the known ERAD complex. As a result, our data suggest that the membrane proteins of SELMA, the derlin proteins, are linked to the soluble sCdc48 complex via the UBX protein sUBX. This is similar to the ERAD machinery whereas the additional SELMA components, sPUB und a second Cdc48 copy might indicate the influence of functional constraints in developing a translocation machinery from ERAD-related factors. In addition, we show for the first time that a rhomboid protease is a central interaction partner of the membrane proteins of the SELMA system in complex plastids.

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

    PubMed

    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.

  7. Folding and self-assembly of a small protein complex

    PubMed Central

    Sieradzan, Adam K.; Liwo, Adam; Hansmann, Ulrich H.E.

    2012-01-01

    The synthetic homotetrameric ββα (BBAT1) protein possesses a stable quaternary structure with a ββα fold. Because of its small size (a total of 84 residues), the homotetramer is an excellent model system with which to study the self-assembly and protein-protein interactions. We find from replica exchange molecular dynamics simulations with the coarse-grain UNRES force field that the folding and association pathway consists of three well-separated steps, where that association to a tetramer precedes and facilitates folding of the four chains. At room temperature the tetramer exists in an ensemble of diverse structures. The crystal structure becomes energetically favored only when the molecule is put in a dense and crystal-like environment. The observed picture of folding promoted by association may mirror the mechanism according to which intrinsically unfolded proteins assume their functional structure. PMID:24039552

  8. Crystallization of bFGF-DNA Aptamer Complexes Using a Sparse Matrix Designed for Protein-Nucleic Acid Complexes

    NASA Technical Reports Server (NTRS)

    Cannone, Jaime J.; Barnes, Cindy L.; Achari, Aniruddha; Kundrot, Craig E.; Whitaker, Ann F. (Technical Monitor)

    2001-01-01

    The Sparse Matrix approach for obtaining lead crystallization conditions has proven to be very fruitful for the crystallization of proteins and nucleic acids. Here we report a Sparse Matrix developed specifically for the crystallization of protein-DNA complexes. This method is rapid and economical, typically requiring 2.5 mg of complex to test 48 conditions. The method was originally developed to crystallize basic fibroblast growth factor (bFGF) complexed with DNA sequences identified through in vitro selection, or SELEX, methods. Two DNA aptamers that bind with approximately nanomolar affinity and inhibit the angiogenic properties of bFGF were selected for co-crystallization. The Sparse Matrix produced lead crystallization conditions for both bFGF-DNA complexes.

  9. Connecting mononuclear dysprosium single-molecule magnets to form dinuclear complexes via in situ ligand oxidation.

    PubMed

    Yutronkie, Nathan J; Kühne, Irina A; Korobkov, Ilia; Brusso, Jaclyn L; Murugesu, Muralee

    2016-01-14

    A Dy2 complex, exhibiting SMM behaviour, and its Y analogue were prepared via in situ oxidation of Py2TTA, a pincer type ligand, followed by dimerisation. This unique metal complexation and subsequent dimerization were followed by solution NMR studies. PMID:26567706

  10. Fluid-dynamic computations on a connection machine - Preliminary timings and complex boundary conditions

    NASA Astrophysics Data System (ADS)

    Oran, Elaine S.; Boris, Jay P.; Brown, Eugene F.

    1990-01-01

    This paper describes the conversion and application of the explicit, time-dependent, fourth-order, phase-accurate, variable-grid flux-corrected transport module, LCPFCT, to the Connection Machine, a fine-grained SIMD parallel processor. Models developed are as similar to the production Cray codes as possible and include a variety of different realistic boundary conditions. Timing comparisons show that a 16K-processor Connection Machine allows computations at speeds up to a factor of seven faster than obtained on a Cray YMP for a functionally equivalent optimized, three-dimensional code. Test calculations of a two-dimensional exploding shock and a three-dimensional helically perturbed jet are described and discussed briefly.

  11. Two-dimensional mesh-connected parallel processor with complex processing elements

    NASA Astrophysics Data System (ADS)

    Chen, Chaoyang; Shen, Xubang; Wang, Zhong; Sang, Hongshi

    2001-09-01

    LS MPP is a massively parallel processor .It has fine-grained parallelism with up to 4096 processing elements arranged in a SIMD architecture .The processing elements are arranged in 64x64 two-dimensional mesh-connected array for low-level image processing .In this paper, the system architecture ,the components of processing element ,array controller ,memory organization of LS MPP processor are described .In the final ,we have discussed the performance of LS MPP.

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

    PubMed

    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.

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

    NASA Astrophysics Data System (ADS)

    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.

  14. Students' Learning with the Connected Chemistry (CC1) Curriculum: Navigating the Complexities of the Particulate World

    NASA Astrophysics Data System (ADS)

    Levy, Sharona T.; Wilensky, Uri

    2009-06-01

    The focus of this study is students' learning with a Connected Chemistry unit, CC1 (denotes Connected Chemistry, chapter 1), a computer-based environment for learning the topics of gas laws and kinetic molecular theory in chemistry (Levy and Wilensky 2009). An investigation was conducted into high-school students' learning with Connected Chemistry, based on a conceptual framework that highlights several forms of access to understanding the system (submicro, macro, mathematical, experiential) and bidirectional transitions among these forms, anchored at the common and experienced level, the macro-level. Results show a strong effect size for embedded assessment and a medium effect size regarding pre-post-test questionnaires. Stronger effects are seen for understanding the submicroscopic level and bridging between it and the macroscopic level. More than half the students succeeded in constructing the equations describing the gas laws. Significant shifts were found in students' epistemologies of models: understanding models as representations rather than replicas of reality and as providing multiple perspectives. Students' learning is discussed with respect to the conceptual framework and the benefits of assessment of learning using a fine-tuned profile and further directions for research are proposed.

  15. Common and distinctive localization patterns of Crumbs polarity complex proteins in the mammalian eye

    PubMed Central

    Kim, Jin Young; Song, Ji Yun; Karnam, Santi; Park, Jun Young; Lee, Jamie JH; Kim, Seonhee; Cho, Seo-Hee

    2016-01-01

    Crumbs polarity complex proteins are essential for cellular and tissue polarity, and for adhesion of epithelial cells. In epithelial tissues deletion of any of three core proteins disrupts localization of the other proteins, indicating structural and functional interdependence among core components. Despite previous studies of function and co-localization that illustrated the properties that these proteins share, it is not known whether an individual component of the complex plays a distinct role in a unique cellular and developmental context. In order to investigate this question, we primarily used confocal imaging to determine the expression and subcellular localization of the core Crumbs polarity complex proteins during ocular development. Here we show that in developing ocular tissues core Crumbs polarity complex proteins, Crb, Pals1 and Patj, generally appear in an overlapping pattern with some exceptions. All three core complex proteins localize to the apical junction of the retinal and lens epithelia. Pals1 is also localized in the Golgi of the retinal cells and Patj localizes to the nuclei of the apically located subset of progenitor cells. These findings suggest that core Crumbs polarity complex proteins exert common and independent functions depending on cellular context. PMID:25636444

  16. PepX: a structural database of non-redundant protein-peptide complexes.

    PubMed

    Vanhee, Peter; Reumers, Joke; Stricher, Francois; Baeten, Lies; Serrano, Luis; Schymkowitz, Joost; Rousseau, Frederic

    2010-01-01

    Although protein-peptide interactions are estimated to constitute up to 40% of all protein interactions, relatively little information is available for the structural details of these interactions. Peptide-mediated interactions are a prime target for drug design because they are predominantly present in signaling and regulatory networks. A reliable data set of nonredundant protein-peptide complexes is indispensable as a basis for modeling and design, but current data sets for protein-peptide interactions are often biased towards specific types of interactions or are limited to interactions with small ligands. In PepX (http://pepx.switchlab.org), we have designed an unbiased and exhaustive data set of all protein-peptide complexes available in the Protein Data Bank with peptide lengths up to 35 residues. In addition, these complexes have been clustered based on their binding interfaces rather than sequence homology, providing a set of structurally diverse protein-peptide interactions. The final data set contains 505 unique protein-peptide interface clusters from 1431 complexes. Thorough annotation of each complex with both biological and structural information facilitates searching for and browsing through individual complexes and clusters. Moreover, we provide an additional source of data for peptide design by annotating peptides with naturally occurring backbone variations using fragment clusters from the BriX database.

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

    PubMed

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

    2015-07-28

    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.

  18. Single-molecule observation of protein folding in symmetric GroEL-(GroES)2 complexes.

    PubMed

    Takei, Yodai; Iizuka, Ryo; Ueno, Taro; Funatsu, Takashi

    2012-11-30

    The chaperonin, GroEL, is an essential molecular chaperone that mediates protein folding together with its cofactor, GroES, in Escherichia coli. It is widely believed that the two rings of GroEL alternate between the folding active state coupled to GroES binding during the reaction cycle. In other words, an asymmetric GroEL-GroES complex (the bullet-shaped complex) is formed throughout the cycle, whereas a symmetric GroEL-(GroES)(2) complex (the football-shaped complex) is not formed. We have recently shown that the football-shaped complex coexists with the bullet-shaped complex during the reaction cycle. However, how protein folding proceeds in the football-shaped complex remains poorly understood. Here, we used GFP as a substrate to visualize protein folding in the football-shaped complex by single-molecule fluorescence techniques. We directly showed that GFP folding occurs in both rings of the football-shaped complex. Remarkably, the folding was a sequential two-step reaction, and the kinetics were in excellent agreement with those in the bullet-shaped complex. These results demonstrate that the same reactions take place independently in both rings of the football-shaped complex to facilitate protein folding. PMID:23048033

  19. Structural basis of pyrimidine specificity in the MS2 RNA hairpin-coat-protein complex.

    PubMed Central

    Grahn, E; Moss, T; Helgstrand, C; Fridborg, K; Sundaram, M; Tars, K; Lago, H; Stonehouse, N J; Davis, D R; Stockley, P G; Liljas, L

    2001-01-01

    We have determined the X-ray structures of six MS2 RNA hairpin-coat-protein complexes having five different substitutions at the hairpin loop base -5. This is a uracil in the wild-type hairpin and contacts the coat protein both by stacking on to a tyrosine side chain and by hydrogen bonding to an asparagine side chain. The RNA consensus sequence derived from coat protein binding studies with natural sequence variants suggested that the -5 base needs to be a pyrimidine for strong binding. The five -5 substituents used in this study were 5-bromouracil, pyrimidin-2-one, 2-thiouracil, adenine, and guanine. The structure of the 5-bromouracil complex was determined to 2.2 A resolution, which is the highest to date for any MS2 RNA-protein complex. All the complexes presented here show very similar conformations, despite variation in affinity in solution. The results suggest that the stacking of the -5 base on to the tyrosine side chain is the most important driving force for complex formation. A number of hydrogen bonds that are present in the wild-type complex are not crucial for binding, as they are missing in one or more of the complexes. The results also reveal the flexibility of this RNA-protein interface, with respect to functional group variation, and may be generally applicable to other RNA-protein complexes. PMID:11720290

  20. Dissociation of a Dynamic Protein Complex Studied by All-Atom Molecular Simulations.

    PubMed

    Zhang, Liqun; Borthakur, Susmita; Buck, Matthias

    2016-02-23

    The process of protein complex dissociation remains to be understood at the atomic level of detail. Computers now allow microsecond timescale molecular-dynamics simulations, which make the visualization of such processes possible. Here, we investigated the dissociation process of the EphA2-SHIP2 SAM-SAM domain heterodimer complex using unrestrained all-atom molecular-dynamics simulations. Previous studies on this system have shown that alternate configurations are sampled, that their interconversion can be fast, and that the complex is dynamic by nature. Starting from different NMR-derived structures, mutants were designed to stabilize a subset of configurations by swapping ion pairs across the protein-protein interface. We focused on two mutants, K956D/D1235K and R957D/D1223R, with attenuated binding affinity compared with the wild-type proteins. In contrast to calculations on the wild-type complexes, the majority of simulations of these mutants showed protein dissociation within 2.4 μs. During the separation process, we observed domain rotation and pivoting as well as a translation and simultaneous rolling, typically to alternate and weaker binding interfaces. Several unsuccessful recapturing attempts occurred once the domains were moderately separated. An analysis of protein solvation suggests that the dissociation process correlates with a progressive loss of protein-protein contacts. Furthermore, an evaluation of internal protein dynamics using quasi-harmonic and order parameter analyses indicates that changes in protein internal motions are expected to contribute significantly to the thermodynamics of protein dissociation. Considering protein association as the reverse of the separation process, the initial role of charged/polar interactions is emphasized, followed by changes in protein and solvent dynamics. The trajectories show that protein separation does not follow a single distinct pathway, but suggest that the mechanism of dissociation is common in

  1. Crucial HSP70 co–chaperone complex unlocks metazoan protein disaggregation

    PubMed Central

    Nillegoda, Nadinath B.; Kirstein, Janine; Szlachcic, Anna; Berynskyy, Mykhaylo; Stank, Antonia; Stengel, Florian; Arnsburg, Kristin; Gao, Xuechao; Scior, Annika; Aebersold, Ruedi; Guilbride, D. Lys; Wade, Rebecca C.; Morimoto, Richard I.; Mayer, Matthias P.; Bukau, Bernd

    2016-01-01

    Protein aggregates are the hallmark of stressed and ageing cells, and characterize several pathophysiological states1,2. Healthy metazoan cells effectively eliminate intracellular protein aggregates3,4, indicating that efficient disaggregation and/or degradation mechanisms exist. However, metazoans lack the key heat-shock protein disaggregase HSP100 of non-metazoan HSP70-dependent protein disaggregation systems5,6, and the human HSP70 system alone, even with the crucial HSP110 nucleotide exchange factor, has poor disaggregation activity in vitro4,7. This unresolved conundrum is central to protein quality control biology. Here we show that synergic cooperation between complexed J-protein co-chaperones of classes A and B unleashes highly efficient protein disaggregation activity in human and nematode HSP70 systems. Metazoan mixed-class J-protein complexes are transient, involve complementary charged regions conserved in the J-domains and carboxy-terminal domains of each J-protein class, and are flexible with respect to subunit composition. Complex formation allows J-proteins to initiate transient higher order chaperone structures involving HSP70 and interacting nucleotide exchange factors. A network of cooperative class A and B J-protein interactions therefore provides the metazoan HSP70 machinery with powerful, flexible, and finely regulatable disaggregase activity and a further level of regulation crucial for cellular protein quality control. PMID:26245380

  2. The low density lipoprotein receptor-related protein/alpha2-macroglobulin receptor is a receptor for connective tissue growth factor.

    PubMed

    Segarini, P R; Nesbitt, J E; Li, D; Hays, L G; Yates, J R; Carmichael, D F

    2001-11-01

    Connective tissue growth factor (CTGF) expression is regulated by transforming growth factor-beta (TGF-beta) and strong up-regulation occurs during wound healing; in situ hybridization data indicate that there are high levels of CTGF expression in fibrotic lesions. Recently the binding parameters of CTGF to both high and lower affinity cell surface binding components have been characterized. Affinity cross-linking and SDS-polyacrylamide gel electrophoresis analysis demonstrated the binding of CTGF to a cell surface protein with a mass of approximately 620 kDa. We report here the purification of this protein by affinity chromatography on CTGF coupled to Sepharose and sequence information obtained by mass spectroscopy. The binding protein was identified as the multiligand receptor, low density lipoprotein receptor-related protein/alpha2-macroglobulin receptor (LRP). The identification of LRP as a receptor for CTGF was validated by several studies: 1) binding competition with many ligands that bind to LRP, including receptor-associated protein; 2) immunoprecipitation of CTGF-receptor complex with LRP antibodies; and 3) cells that are genetically deficient for LRP were unable to bind CTGF. Last, CTGF is rapidly internalized and degraded and this process is LRP-dependent. In summary, our data indicate that LRP is a receptor for CTGF, and may play an important role in mediating CTGF biology.

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

    NASA Astrophysics Data System (ADS)

    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.

  4. Protein Crystal Eco R1 Endonulease-DNA Complex

    NASA Technical Reports Server (NTRS)

    1998-01-01

    Type II restriction enzymes, such as Eco R1 endonulease, present a unique advantage for the study of sequence-specific recognition because they leave a record of where they have been in the form of the cleaved ends of the DNA sites where they were bound. The differential behavior of a sequence -specific protein at sites of differing base sequence is the essence of the sequence-specificity; the core question is how do these proteins discriminate between different DNA sequences especially when the two sequences are very similar. Principal Investigator: Dan Carter/New Century Pharmaceuticals

  5. Prediction of protein complexes using empirical free energy functions.

    PubMed Central

    Weng, Z.; Vajda, S.; Delisi, C.

    1996-01-01

    A long sought goal in the physical chemistry of macromolecular structure, and one directly relevant to understanding the molecular basis of biological recognition, is predicting the geometry of bimolecular complexes from the geometries of their free monomers. Even when the monomers remain relatively unchanged by complex formation, prediction has been difficult because the free energies of alternative conformations of the complex have been difficult to evaluate quickly and accurately. This has forced the use of incomplete target functions, which typically do no better than to provide tens of possible complexes with no way of choosing between them. Here we present a general framework for empirical free energy evaluation and report calculations, based on a relatively complete and easily executable free energy function, that indicate that the structures of complexes can be predicted accurately from the structures of monomers, including close sequence homologues. The calculations also suggest that the binding free energies themselves may be predicted with reasonable accuracy. The method is compared to an alternative formulation that has also been applied recently to the same data set. Both approaches promise to open new opportunities in macromolecular design and specificity modification. PMID:8845751

  6. Modifications in structure and interaction of nanoparticle-protein-surfactant complexes in electrolyte solution

    NASA Astrophysics Data System (ADS)

    Mehan, Sumit; Kumar, S.; Aswal, V. K.; Schweins, R.

    2016-05-01

    SANS experiments of three-component system of anionic silica nanoparticles, anionic BSA protein and anionic SDS surfactants have been carried out without and with electrolyte in aqueous solution. In both the cases, the interaction of surfactant with protein results in formation of bead-necklace structure of protein-surfactant complexes in solution. These protein-surfactant complexes interact very differently with nanoparticles in absence and presence of electrolyte. In absence of electrolyte, nanoparticles remain in dispersed phase in solution, whereas with the addition of electrolyte the nanoparticles fractal aggregates are formed. SANS describes the phase behavior to be governed by competition of electrostatic and depletion interactions among the components solution.

  7. The neuronal proteins CIPP, Cypin and IRSp53 form a tripartite complex mediated by PDZ and SH3 domains.

    PubMed

    Barilari, Manuela; Dente, Luciana

    2010-10-01

    Here we report the dissection of a tripartite complex formed by CIPP (channel-interacting PDZ protein), IRSp53 (insulin receptor tyrosine kinase substrate protein) and Cypin (cytosolic PSD-95 interactor) in cultured cells. The three proteins are expressed in similar neuronal districts, where CIPP binds to different membrane channels and receptors, IRSp53 regulates the morphogenesis of actin-rich dendritic spines, and Cypin promotes dendrite branching and patterning by binding to tubulin heterodimers. We observed that the interaction among the three proteins is mediated by small binding domains: CIPP works as a bridge, linking the carboxy-termini of IRSp53 and Cypin with its PDZ domains; IRSp53 connects Cypin, through an unusual SH3-mediated association, which can be impaired by substituting two crucial positively charged residues of Cypin. The observation that the three engineered proteins co-localize in the cytoplasm, and at the tip of induced neurites in neuronal cells, raises the interesting possibility that they work together in the formation of neuronal protrusions.

  8. Novel thioredoxin-like proteins are components of a protein complex coating the cortical microtubules of Toxoplasma gondii.

    PubMed

    Liu, Jun; Wetzel, Laura; Zhang, Ying; Nagayasu, Eiji; Ems-McClung, Stephanie; Florens, Laurence; Hu, Ke

    2013-12-01

    Microtubules are versatile biopolymers that support numerous vital cellular functions in eukaryotes. The specific properties of microtubules are dependent on distinct microtubule-associated proteins, as the tubulin subunits and microtubule structure are exceptionally conserved. Highly specialized microtubule-containing assemblies are often found in protists, which are rich sources for novel microtubule-associated proteins. A protozoan parasite, Toxoplasma gondii, possesses several distinct tubulin-containing structures, including 22 microtubules closely associated with the cortical membrane. Early ultrastructural studies have shown that the cortical microtubules are heavily decorated with associating proteins. However, little is known about the identities of these proteins. Here, we report the discovery of a novel protein, TrxL1 (for Thioredoxin-Like protein 1), and an associating complex that coats the cortical microtubules. TrxL1 contains a thioredoxin-like fold. To visualize its localization in live parasites by fluorescence, we replaced the endogenous TrxL1 gene with an mEmeraldFP-TrxL1 fusion gene. Structured illumination-based superresolution imaging of this parasite line produced a detailed view of the microtubule cytoskeleton. Despite its stable association with the cortical microtubules in the parasite, TrxL1 does not seem to bind to microtubules directly. Coimmunoprecipitation experiments showed that TrxL1 associates with a protein complex containing SPM1, a previously reported microtubule-associated protein in T. gondii. We also found that SPM1 recruits TrxL1 to the cortical microtubules. Besides SPM1, several other novel proteins are found in the TrxL1-containing complex, including TrxL2, a close homolog of TrxL1. Thus, our results reveal for the first time a microtubule-associated complex in T. gondii.

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

    PubMed

    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.

  10. Bcl-2 proteins and calcium signaling: complexity beneath the surface.

    PubMed

    Vervliet, T; Parys, J B; Bultynck, G

    2016-09-29

    Antiapoptotic Bcl-2-family members are well known for their 'mitochondrial' functions as critical neutralizers of proapoptotic Bcl-2-family members, including the executioner multidomain proteins Bax and Bak and the BH3-only proteins. It has been clear for more than 20 years that Bcl-2 proteins can impact intracellular Ca(2+) homeostasis and dynamics. Moreover, altered Ca(2+) signaling is increasingly linked to oncogenic behavior. Specifically targeting the Ca(2+)-signaling machinery may thus prove to be a valuable strategy for cancer treatment. Over 10 years ago a major controversy was recognized concerning whether or not Bcl-2 proteins exerted their antiapoptotic functions via Ca(2+) signaling through lowering the filling state of the endoplasmic reticulum (ER) Ca(2+) stores or by suppressing Ca(2+) release from the ER without affecting the filling state of this Ca(2+) store. Further research from different laboratories indicated a wide variety of mechanisms by which Bcl-2-family members can impact Ca(2+) signaling. In this review, we propose that antiapoptotic Bcl-2-family members are multimodal regulators of intracellular Ca(2+)-signaling events in cell survival and cell death. We will discuss how different Bcl-2-family members impact cell survival and cell death by regulating Ca(2+) transport systems at the ER, mitochondria and plasma membrane and by impacting the organization of organelles and how these insights can be exploited for causing cell death in cancer cells. Finally, we propose that the existing controversy reflects the diversity of links between Bcl-2 proteins and Ca(2+) signaling, as certainly not all targets or mechanisms will be operative in every cell type and every condition.

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

    PubMed

    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.

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

    PubMed

    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

  13. Sequence co-evolution gives 3D contacts and structures of protein complexes

    PubMed Central

    Hopf, Thomas A; Schärfe, Charlotta P I; Rodrigues, João P G L M; Green, Anna G; Kohlbacher, Oliver; Sander, Chris; Bonvin, Alexandre M J J; Marks, Debora S

    2014-01-01

    Protein–protein interactions are fundamental to many biological processes. Experimental screens have identified tens of thousands of interactions, and structural biology has provided detailed functional insight for select 3D protein complexes. An alternative rich source of information about protein interactions is the evolutionary sequence record. Building on earlier work, we show that analysis of correlated evolutionary sequence changes across proteins identifies residues that are close in space with sufficient accuracy to determine the three-dimensional structure of the protein complexes. We evaluate prediction performance in blinded tests on 76 complexes of known 3D structure, predict protein–protein contacts in 32 complexes of unknown structure, and demonstrate how evolutionary couplings can be used to distinguish between interacting and non-interacting protein pairs in a large complex. With the current growth of sequences, we expect that the method can be generalized to genome-wide elucidation of protein–protein interaction networks and used for interaction predictions at residue resolution. DOI: http://dx.doi.org/10.7554/eLife.03430.001 PMID:25255213

  14. Resveratrol upregulates Egr-1 expression and activity involving extracellular signal-regulated protein kinase and ternary complex factors

    SciTech Connect

    Rössler, Oliver G.; Glatzel, Daniel; Thiel, Gerald

    2015-03-01

    Many intracellular functions have been attributed to resveratrol, a polyphenolic phytoalexin found in grapes and in other plants. Here, we show that resveratrol induces the expression of the transcription factor Egr-1 in human embryonic kidney cells. Using a chromosomally embedded Egr-1-responsive reporter gene, we show that the Egr-1 activity was significantly elevated in resveratrol-treated cells, indicating that the newly synthesized Egr-1 protein was biologically active. Stimulus-transcription coupling leading to the resveratrol-induced upregulation of Egr-1 expression and activity requires the protein kinases Raf and extracellular signal-regulated protein kinase ERK, while MAP kinase phosphatase-1 functions as a nuclear shut-off device that interrupts the signaling cascade connecting resveratrol stimulation with enhanced Egr-1 expression. On the transcriptional level, Elk-1, a key transcriptional regulator of serum response element-driven gene transcription, connects the intracellular signaling cascade elicited by resveratrol with transcription of the Egr-1 gene. These data were corroborated by the observation that stimulation of the cells with resveratrol increased the transcriptional activation potential of Elk-1. The SRE as well as the GC-rich DNA binding site of Egr-1 function as resveratrol-responsive elements. Thus, resveratrol regulates gene transcription via activation of the stimulus-regulated protein kinases Raf and ERK and the stimulus-responsive transcription factors TCF and Egr-1. - Highlights: • The plant polyphenol resveratrol upregulates Egr-1 expression and activity. • The stimulation of Egr-1 requires the protein kinases ERK and Raf. • Resveratrol treatment upregulates the transcriptional activation potential of Elk-1. • Resveratrol-induced stimulation of Egr-1 requires ternary complex factors. • Two distinct resveratrol-responsive elements were identified.

  15. Remotely sensed image classification by complex network eigenvalue and connected degree.

    PubMed

    Xu, Mengxi; Wei, Chenglin

    2012-01-01

    It is a well-known problem of remotely sensed images classification due to its complexity. This paper proposes a remotely sensed image classification method based on weighted complex network clustering using the traditional K-means clustering algorithm. First, the degree of complex network and clustering coefficient of weighted feature are used to extract the features of the remote sensing image. Then, the integrated features of remote sensing image are combined to be used as the basis of classification. Finally, K-means algorithm is used to classify the remotely sensed images. The advantage of the proposed classification method lies in obtaining better clustering centers. The experimental results show that the proposed method gives an increase of 8% in accuracy compared with the traditional K-means algorithm and the Iterative Self-Organizing Data Analysis Technique (ISODATA) algorithm.

  16. Combination of MS protein identification and bioassay of chromatographic fractions to identify biologically active substances from complex protein sources.

    PubMed

    Kuromitsu, Sadao; Yokota, Hiroyuki; Hiramoto, Masashi; Yuri, Masatoshi; Naitou, Masanori; Nakamura, Naoto; Kawabata, Shigeki; Kobori, Masato; Katoh, Masao; Furuchi, Kiyoshi; Mita, Haruhisa; Yamada, Tetsuo

    2009-06-01

    Purification of biologically active proteins from complex biological sources is a difficult task, usually requiring large amounts of sample and many separation steps. We found an active substance in a serum response element-dependent luciferase reporter gene bioassay in interstitial cystitis urine that we attempted to purify with column chromatography and the bioassay. With anion-exchange Mono Q and C4 reversed-phase columns, apparently sharp active peaks were obtained. However, more than 20 kinds of proteins were identified from the active fractions with MS, indicating that the purification was not complete. As further purification was difficult, we chose a candidate molecule by means of studying the correlation between MS protein identification scores and bioassay responses of chromatographic fractions near the active peaks. As a result, epidermal growth factor (EGF) was nominated as a candidate molecule among the identified proteins because the elution profile of EGF was consistent with that of the bioassay, and the correlation coefficient of EGF between MS protein identification scores and bioassay responses was the highest among all the identified proteins. With recombinant EGF and anti-EGF and anti-EGF receptor antibodies, EGF was confirmed to be the desired substance in interstitial cystitis urine. This approach required only 20 ml of urine sample and two column chromatographic steps. The combination of MS protein identification and bioassay of chromatographic fractions may be useful for identifying biologically active substances from complex protein sources.

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

    NASA Technical Reports Server (NTRS)

    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.

  18. Immunological changes following protein losing enteropathy after surgery total cavopulmonary connection (TCPC) by cytomics

    NASA Astrophysics Data System (ADS)

    Bocsi, József; Lenz, Dominik; Mittag, Anja; Sauer, Ursula; Wild, Lena; Hess, John; Schranz, Dietmar; Hambsch, Jörg; Schneider, Peter; Tárnok, Attila

    2008-02-01

    Complex immunophenotyping single-cell analysis are essential for systems biology and cytomics. The application of cytomics in immunology and cardiac research and diagnostics is very broad, ranging from the better understanding of the cardiovascular cell biology to the identification of heart function and immune consequences after surgery. TCPC or Fontan-type circulation is an accepted palliative surgery for patients with a functionally univentricular heart. Protein-losing enteropathy (PLE), the enteric loss of proteins, is a potential late complication after TCPC surgery. PLE etiology is poorly understood, but immunological factors seem to play a role. This study was aimed to gain insight into immune phenotype alterations following post-TCPC PLE. Patients were studied during routine follow-up up to 5yrs after surgery, blood samples of TCPC patients without (n=21, age 6.8+/-2.6 years at surgery; mean+/-SD) and with manifest PLE (n=12, age 12.8+/- 4.5 years at sampling) and age matched healthy children (control, n=22, age 8.6+/-2.5 years) were collected. Routine laboratory, immune phenotype and serological parameters were determined. Following PLE the immune phenotype dramatically changed with signs of acute inflammation (increased neutrophil and monocyte count, CRP, IL-8). In contrast, lymphocyte count (NK-cells, αβTCR +CD4 +, αβTCR +CD8 + cells) decreased (p<0.001). The residual T-cells had elevated CD25 and CD69 expression. In PLE-patients unique cell populations with CD3 +αβ/γδTCR - and αβTCR +CD4 -8 - phenotype were present in increased frequencies. Our studies show dramatically altered leukocyte phenotype after PLE in TCPC patients. These alterations resemble to changes in autoimmune diseases. We conclude that autoimmune processes may play a role in etiology and pathophysiology of PLE.

  19. Connecting the Dots: Social Network Structure, Conflict, and Group Cognitive Complexity

    ERIC Educational Resources Information Center

    Curseu, Petru L.; Janssen, Steffie E. A.; Raab, Jorg

    2012-01-01

    The current paper combines arguments from the social capital and group cognition literature to explain two different processes through which communication network structures and intra group conflict influence groups' cognitive complexity (GCC). We test in a sample of 44 groups the mediating role of intra group conflict in the relationship between…

  20. Trauma Adapted Family Connections: Reducing Developmental and Complex Trauma Symptomatology to Prevent Child Abuse and Neglect

    ERIC Educational Resources Information Center

    Collins, Kathryn S.; Strieder, Frederick H.; DePanfilis, Diane; Tabor, Maureen; Clarkson Freeman, Pamela A.; Linde, Linnea; Greenberg, Patty

    2011-01-01

    Families living in urban poverty, enduring chronic and complex traumatic stress, and having difficulty meeting their children's basic needs have significant child maltreatment risk factors. There is a paucity of family focused, trauma-informed evidence-based interventions aimed to alleviate trauma symptomatology, strengthen family functioning, and…

  1. Promoter Decommissioning by the NuRD Chromatin Remodeling Complex Triggers Synaptic Connectivity in the Mammalian Brain

    PubMed Central

    Yamada, Tomoko; Yang, Yue; Hemberg, Martin; Yoshida, Toshimi; Cho, Ha Young; Murphy, J. Patrick; Fioravante, Diasynou; Regehr, Wade G.; Gygi, Steven P.; Georgopoulos, Katia; Bonni, Azad

    2014-01-01

    SUMMARY Precise control of gene expression plays fundamental roles in brain development, but the roles of chromatin regulators in neuronal connectivity have remained poorly understood. We report that depletion of the NuRD complex by in vivo RNAi and conditional knockout of the core NuRD subunit Chd4 profoundly impairs the establishment of granule neuron parallel fiber/Purkinje cell synapses in the rodent cerebellar cortex in vivo. By interfacing genome-wide sequencing of transcripts and ChIP-Seq analyses, we uncover a network of repressed genes and distinct histone modifications at target gene promoters that are developmentally regulated by the NuRD complex in the cerebellum in vivo. Finally, in a targeted in vivo RNAi screen of NuRD target genes, we identify a program of NuRD-repressed genes that operate as critical regulators of presynaptic differentiation in the cerebellar cortex. Our findings define NuRD-dependent promoter decommissioning as a developmentally-regulated programming mechanism that drives synaptic connectivity in the mammalian brain. PMID:24991957

  2. Molecular recognition of DNA-protein complexes: a straightforward method combining scanning force and fluorescence microscopy.

    PubMed

    Sanchez, Humberto; Kanaar, Roland; Wyman, Claire

    2010-06-01

    Combining scanning force and fluorescent microscopy allows simultaneous identification of labeled biomolecules and analysis of their nanometer level architectural arrangement. Fluorescent polystyrene nano-spheres were used as reliable objects for alignment of optical and topographic images. This allowed the precise localization of different fluorescence particles within complex molecular assemblies whose structure was mapped in nanometer detail topography. Our experiments reveal the versatility of this method for analysis of proteins and protein-DNA complexes.

  3. The Hsp90-Dependent Proteome Is Conserved and Enriched for Hub Proteins with High Levels of Protein–Protein Connectivity

    PubMed Central

    Swamy, Krishna B.S.; Yu, Jau-Song; Schuyler, Scott C.; Leu, Jun-Yi

    2014-01-01

    Hsp90 is one of the most abundant and conserved proteins in the cell. Reduced levels or activity of Hsp90 causes defects in many cellular processes and also reveals genetic and nongenetic variation within a population. Despite information about Hsp90 protein–protein interactions, a global view of the Hsp90-regulated proteome in yeast is unavailable. To investigate the degree of dependency of individual yeast proteins on Hsp90, we used the “stable isotope labeling by amino acids in cell culture” method coupled with mass spectrometry to quantify around 4,000 proteins in low-Hsp90 cells. We observed that 904 proteins changed in their abundance by more than 1.5-fold. When compared with the transcriptome of the same population of cells, two-thirds of the misregulated proteins were observed to be affected posttranscriptionally, of which the majority were downregulated. Further analyses indicated that the downregulated proteins are highly conserved and assume central roles in cellular networks with a high number of protein interacting partners, suggesting that Hsp90 buffers genetic and nongenetic variation through regulating protein network hubs. The downregulated proteins were enriched for essential proteins previously not known to be Hsp90-dependent. Finally, we observed that downregulation of transcription factors and mating pathway components by attenuating Hsp90 function led to decreased target gene expression and pheromone response, respectively, providing a direct link between observed proteome regulation and cellular phenotypes. PMID:25316598

  4. Adaptive selection and coevolution at the proteins of the Polycomb repressive complexes in Drosophila.

    PubMed

    Calvo-Martín, J M; Librado, P; Aguadé, M; Papaceit, M; Segarra, C

    2016-02-01

    Polycomb group (PcG) proteins are important epigenetic regulatory proteins that modulate the chromatin state through posttranslational histone modifications. These interacting proteins form multimeric complexes that repress gene expression. Thus, PcG proteins are expected to evolve coordinately, which might be reflected in their phylogenetic trees by concordant episodes of positive selection and by a correlation in evolutionary rates. In order to detect these signals of coevolution, the molecular evolution of 17 genes encoding the subunits of five Polycomb repressive complexes has been analyzed in the Drosophila genus. The observed distribution of divergence differs substantially among and along proteins. Indeed, CAF1 is uniformly conserved, whereas only the established protein domains are conserved in other proteins, such as PHO, PHOL, PSC, PH-P and ASX. Moreover, regions with a low divergence not yet described as protein domains are present, for instance, in SFMBT and SU(Z)12. Maximum likelihood methods indicate an acceleration in the nonsynonymous substitution rate at the lineage ancestral to the obscura group species in most genes encoding subunits of the Pcl-PRC2 complex and in genes Sfmbt, Psc and Kdm2. These methods also allow inferring the action of positive selection in this lineage at genes E(z) and Sfmbt. Finally, the protein interaction network predicted from the complete proteomes of 12 Drosophila species using a coevolutionary approach shows two tight PcG clusters. These clusters include well-established binary interactions among PcG proteins as well as new putative interactions.

  5. Resting state functional connectivity analysis for addiction medicine: From individual loci to complex networks.

    PubMed

    Pariyadath, Vani; Gowin, Joshua L; Stein, Elliot A

    2016-01-01

    Resting state functional connectivity (rsFC) has provided a new and valuable tool for investigating network-level dysfunction in addiction. Following the recent development of a framework of large scale network disruptions, we have been able to arrive at unique insights into craving-related aspects of addiction using rsFC. However, such network-level advancement has thus far eluded our understanding of mesocorticolimbic involvement in addiction. Given the importance of this system in vulnerability and resilience to addiction, understanding mesocorticolimbic dynamics to the same extent could provide critical insights into the disease. To this end, we review here recent studies on addiction that employ rsfC and suggest a new approach, one that combines a novel model for addiction with new experimental techniques as well as participant groups, to accelerate progress in this arena. PMID:26822358

  6. RNA 3'-end mismatch excision by the severe acute respiratory syndrome coronavirus nonstructural protein nsp10/nsp14 exoribonuclease complex.

    PubMed

    Bouvet, Mickaël; Imbert, Isabelle; Subissi, Lorenzo; Gluais, Laure; Canard, Bruno; Decroly, Etienne

    2012-06-12

    The replication/transcription complex of severe acute respiratory syndrome coronavirus is composed of at least 16 nonstructural proteins (nsp1-16) encoded by the ORF-1a/1b. This complex includes replication enzymes commonly found in positive-strand RNA viruses, but also a set of RNA-processing activities unique to some nidoviruses. The nsp14 protein carries both exoribonuclease (ExoN) and (guanine-N7)-methyltransferase (N7-MTase) activities. The nsp14 ExoN activity ensures a yet-uncharacterized function in the virus life cycle and must be regulated to avoid nonspecific RNA degradation. In this work, we show that the association of nsp10 with nsp14 stimulates >35-fold the ExoN activity of the latter while playing no effect on N7-MTase activity. Nsp10 mutants unable to interact with nsp14 are not proficient for ExoN activation. The nsp10/nsp14 complex hydrolyzes double-stranded RNA in a 3' to 5' direction as well as a single mismatched nucleotide at the 3'-end mimicking an erroneous replication product. In contrast, di-, tri-, and longer unpaired ribonucleotide stretches, as well as 3'-modified RNAs, resist nsp10/nsp14-mediated excision. In addition to the activation of nsp16-mediated 2'-O-MTase activity, nsp10 also activates nsp14 in an RNA processing function potentially connected to a replicative mismatch repair mechanism. PMID:22635272

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

    PubMed

    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.

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

    PubMed

    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.

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

    PubMed

    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

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

    PubMed

    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.

  11. Structure of the Get3 targeting factor in complex with its membrane protein cargo

    PubMed Central

    Mateja, Agnieszka; Paduch, Marcin; Chang, Hsin-Yang; Szydlowska, Anna; Kossiakoff, Anthony A.; Hegde, Ramanujan S.; Keenan, Robert J.

    2015-01-01

    Tail-anchored (TA) proteins are a physiologically important class of membrane proteins targeted to the endoplasmic reticulum by the conserved GET pathway. During transit, their hydrophobic transmembrane domains (TMDs) are chaperoned by the cytosolic targeting factor Get3, but the molecular nature of the functional Get3-TA protein targeting complex remains unknown. We reconstituted the physiologic assembly pathway for a functional targeting complex and showed that it comprises a TA protein bound to a Get3 homodimer. Crystal structures of Get3 bound to different TA proteins showed an α-helical TMD occupying a hydrophobic groove that spans the Get3 homodimer. Our data elucidate the mechanism of TA protein recognition and shielding by Get3, and suggest general principles of hydrophobic domain chaperoning by cellular targeting factors. PMID:25745174

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

    NASA Astrophysics Data System (ADS)

    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

  13. Transient HMGB protein interactions with B-DNA duplexes and complexes

    PubMed Central

    Zimmerman, Jeff; Maher, L. James

    2008-01-01

    HMGB proteins are abundant, non-histone proteins in eukaryotic chromatin. HMGB proteins contain one or two conserved “HMG boxes” and can be sequence specific or nonspecific in their DNA binding. HMGB proteins cause strong DNA bending and bind preferentially to deformed DNAs. We wish to understand how HMGB proteins increase the apparent flexibility of non-distorted B-form DNA. We test the hypothesis that HMGB proteins bind transiently, creating an ensemble of distorted DNAs with rapidly-interconverting conformations. We show that binding of B-form DNA by HMGB proteins is both weak and transient under conditions where DNA cyclization is strongly enhanced. We also detect novel complexes in which HMGB proteins simultaneously bind more than one DNA duplex. PMID:18413230

  14. Search for complexity generating chemical transformations by combining connectivity analysis and cascade transformation patterns.

    PubMed

    Nowak, Grazyna; Fic, Grzegorz

    2010-08-23

    Retrosynthetic analysis involved in a backward search for strategic disconnections is still the most powerful strategy, recently advanced by topology-based complexity estimation, for discovering the shortest sequences of transformations and chemical synthesis planning. Therein, we propose an alternative strategy that combines backward and forward search embodied within a mathematical model of generating chemical transformations. The backward reasoning involves a new concept of the strategic bond tree for alternative multibond disconnections of a target molecule. In the forward direction, each combination of the resulted structural fragments is examined for reconstruction of the target structure by means of biomimetic transformation patterns that describe one-pot multibond forming reactions. The algorithm has been implemented into the CSB system, and its performance is illustrated by examples of published complex molecule syntheses for comparison and analysis. This paper describes the strategy for discovering the shortest synthetic pathways based on the multibond forming cascade transformations for application in synthesis design and generating synthetically accessible product libraries.

  15. Uncoupling proteins: a complex journey to function discovery.

    PubMed

    Cioffi, Federica; Senese, Rosalba; de Lange, Pieter; Goglia, Fernando; Lanni, Antonia; Lombardi, Assunta

    2009-01-01

    Since their discovery, uncoupling proteins have aroused great interest due to the crucial importance of energy-dissipating system for cellular physiology. The uncoupling effect and the physiological role of UCP1 (the first-described uncoupling protein) are well established. However, the reactions catalyzed by UCP1 homologues (UCPs), and their physiological roles are still under debate, with the literature containing contrasting results. Current hypothesis propose several physiological functions for novel UCPs, such as: (i) attenuation of reactive oxygen species production and protection against oxidative damage, (ii) thermogenic function, although UCPs do not generally seem to affect thermogenesis, UCP3 can be thermogenic under certain conditions, (iii) involvement in fatty acid handling and/or transport, although recent experimental evidence argues against the previously hypothesized role for UCPs in the export of fatty acid anions, (iv) fatty acid hydroperoxide export, although this function, due to the paucity of the experimental evidence, remains hypothetical, (v) Ca(2+) uptake, although results for and against a role in Ca(2+) uptake are still emerging, (vi) a signaling role in pancreatic beta cells, where it attenuates glucose-induced insulin secretion. From the above, it is evident that more research will be needed to establish universally accepted functions for UCPs.

  16. Entangled maximal mixings in UPMNS=Ul†Uν, and a connection to complex mass textures

    NASA Astrophysics Data System (ADS)

    Niehage, Svenja; Winter, Walter

    2008-07-01

    We discuss two different configurations of UPMNS=Uℓ†Uν with maximal mixings in both Uℓ and Uν. The nonmaximal mixing angles are assumed to be small, which means that they can be expanded in. Since we are particularly interested in the implications for CP violation, we fully take into account complex phases. We demonstrate that one possibility leads to intrinsically large sin⁡22θ13 and strong deviations from maximal mixings. The other possibility is generically close to tribimaximal mixing, and allows for large CP violation. We demonstrate how the determination of the θ23 octant and the precision measurement of δCP could discriminate among different qualitative cases. In order to constrain the unphysical and observable phases even further, we relate our configurations to complex mass matrix textures. In particular, we focus on phase patterns which could be generated by powers of a single complex quantity η≃θCexp⁡(iΦ), which can be motivated by Froggatt-Nielsen-like models. For example, it turns out that in all of the discussed cases, one of the Majorana phases is proportional to Φ to leading order. In the entire study, we encounter three different classes of sum rules, which we systematically classify.

  17. An informatic framework for decoding protein complexes by top-down mass spectrometry

    PubMed Central

    Skinner, Owen S.; Havugimana, Pierre C.; Haverland, Nicole A.; Fornelli, Luca; Early, Bryan P.; Greer, Joseph B.; Fellers, Ryan T.; Durbin, Kenneth R.; Do Vale, Luis H. F.; Melani, Rafael D.; Seckler, Henrique S.; Nelp, Micah T.; Belov, Mikhail E.; Horning, Stevan R.; Makarov, Alexander A.; LeDuc, Richard D.; Bandarian, Vahe; Compton, Philip D.; Kelleher, Neil L.

    2015-01-01

    Efforts to map the human protein interactome have resulted in information about hundreds to thousands of multi-protein assemblies housed in public repositories, but the molecular characterization and stoichiometry of their protein subunits remains largely unknown. Here, we combined the CORUM and UniProt databases to create candidates for an error-tolerant search engine designed for hierarchical top-down analyses, identification, and scoring of multi-proteoform complexes by native mass spectrometry. PMID:26780093

  18. Binding Linkage in a Telomere DNA–Protein Complex at the Ends of Oxytricha nova Chromosomes

    PubMed Central

    Buczek, Pawel; Orr, Rochelle S.; Pyper, Sean R.; Shum, Mili; Ota, Emily Kimmel Irene; Gerum, Shawn E.; Horvath, Martin P.

    2005-01-01

    Alpha and beta protein subunits of the telomere end binding protein from Oxytricha nova (OnTEBP) combine with telomere single strand DNA to form a protective cap at the ends of chromosomes. We tested how protein–protein interactions seen in the co-crystal structure relate to DNA binding through use of fusion proteins engineered as different combinations of domains and subunits derived from OnTEBP. Joining alpha and beta resulted in a protein that bound single strand telomere DNA with high affinity (KD-DNA=1.4 nM). Another fusion protein, constructed without the C-terminal protein–protein interaction domain of alpha, bound DNA with 200-fold diminished affinity (KD-DNA=290 nM) even though the DNA-binding domains of alpha and beta were joined through a peptide linker. Adding back the alpha C-terminal domain as a separate protein restored high-affinity DNA binding. The binding behaviors of these fusion proteins and the native protein subunits are consistent with cooperative linkage between protein-association and DNA-binding equilibria. Linking DNA–protein stability to protein–protein contacts at a remote site may provide a trigger point for DNA–protein disassembly during telomere replication when the single strand telomere DNA must exchange between a very stable OnTEBP complex and telomerase. PMID:15967465

  19. Multiple barriers in forced rupture of protein complexes

    NASA Astrophysics Data System (ADS)

    Hyeon, Changbong; Thirumalai, D.

    2012-08-01

    Curvatures in the most probable rupture force (f*) versus log-loading rate (log rf) observed in dynamic force spectroscopy (DFS) on biomolecular complexes are interpreted using a one-dimensional free energy profile with multiple barriers or a single barrier with force-dependent transition state. Here, we provide a criterion to select one scenario over another. If the rupture dynamics occurs by crossing a single barrier in a physical free energy profile describing unbinding, the exponent ν, from (1 - f*/fc)1/ν ˜ (log rf) with fc being a critical force in the absence of force, is restricted to 0.5 ⩽ ν ⩽ 1. For biotin-ligand complexes and leukocyte-associated antigen-1 bound to intercellular adhesion molecules, which display large curvature in the DFS data, fits to experimental data yield ν < 0.5, suggesting that if ligand unbinding is assumed to proceed along one-dimensional pulling coordinate, the dynamics should occur in a energy landscape with multiple-barriers.

  20. Identification of protein complexes in Escherichia coli using sequential peptide affinity purification in combination with tandem mass spectrometry.

    PubMed

    Babu, Mohan; Kagan, Olga; Guo, Hongbo; Greenblatt, Jack; Emili, Andrew

    2012-01-01

    -scale cultures. Tandem mass spectrometry is then used to identify the stably co-purifying proteins with high sensitivity (low nanogram detection limits). Here, we describe detailed step-by-step procedures we commonly use for systematic protein tagging, purification and mass spectrometry-based analysis of soluble protein complexes from E. coli, which can be scaled up and potentially tailored to other bacterial species, including certain opportunistic pathogens that are amenable to recombineering. The resulting physical interactions can often reveal interesting unexpected components and connections suggesting novel mechanistic links. Integration of the PPI data with alternate molecular association data such as genetic (gene-gene) interactions and genomic-context (GC) predictions can facilitate elucidation of the global molecular organization of multi-protein complexes within biological pathways. The networks generated for E. coli can be used to gain insight into the functional architecture of orthologous gene products in other microbes for which functional annotations are currently lacking.

  1. Intermolecular detergent-membrane protein noes for the characterization of the dynamics of membrane protein-detergent complexes.

    PubMed

    Eichmann, Cédric; Orts, Julien; Tzitzilonis, Christos; Vögeli, Beat; Smrt, Sean; Lorieau, Justin; Riek, Roland

    2014-12-11

    The interaction between membrane proteins and lipids or lipid mimetics such as detergents is key for the three-dimensional structure and dynamics of membrane proteins. In NMR-based structural studies of membrane proteins, qualitative analysis of intermolecular nuclear Overhauser enhancements (NOEs) or paramagnetic resonance enhancement are used in general to identify the transmembrane segments of a membrane protein. Here, we employed a quantitative characterization of intermolecular NOEs between (1)H of the detergent and (1)H(N) of (2)H-perdeuterated, (15)N-labeled α-helical membrane protein-detergent complexes following the exact NOE (eNOE) approach. Structural considerations suggest that these intermolecular NOEs should show a helical-wheel-type behavior along a transmembrane helix or a membrane-attached helix within a membrane protein as experimentally demonstrated for the complete influenza hemagglutinin fusion domain HAfp23. The partial absence of such a NOE pattern along the amino acid sequence as shown for a truncated variant of HAfp23 and for the Escherichia coli inner membrane protein YidH indicates the presence of large tertiary structure fluctuations such as an opening between helices or the presence of large rotational dynamics of the helices. Detergent-protein NOEs thus appear to be a straightforward probe for a qualitative characterization of structural and dynamical properties of membrane proteins embedded in detergent micelles.

  2. Visualizing Proteins and Macromolecular Complexes by Negative Stain EM: from Grid Preparation to Image Acquisition

    PubMed Central

    Booth, David S.; Avila-Sakar, Agustin; Cheng, Yifan

    2011-01-01

    Single particle electron microscopy (EM), of both negative stained or frozen hydrated biological samples, has become a versatile tool in structural biology 1. In recent years, this method has achieved great success in studying structures of proteins and macromolecular complexes 2, 3. Compared with electron cryomicroscopy (cryoEM), in which frozen hydrated protein samples are embedded in a thin layer of vitreous ice 4, negative staining is a simpler sample preparation method in which protein samples are embedded in a thin layer of dried heavy metal salt to increase specimen contrast 5. The enhanced contrast of negative stain EM allows examination of relatively small biological samples. In addition to determining three-dimensional (3D) structure of purified proteins or protein complexes 6, this method can be used for much broader purposes. For example, negative stain EM can be easily used to visualize purified protein samples, obtaining information such as homogeneity/heterogeneity of the sample, formation of protein complexes or large assemblies, or simply to evaluate the quality of a protein preparation. In this video article, we present a complete protocol for using an EM to observe negatively stained protein sample, from preparing carbon coated grids for negative stain EM to acquiring images of negatively stained sample in an electron microscope operated at 120kV accelerating voltage. These protocols have been used in our laboratory routinely and can be easily followed by novice users. PMID:22215030

  3. Molecular counting by photobleaching in protein complexes with many subunits: best practices and application to the cellulose synthesis complex

    PubMed Central

    Chen, Yalei; Deffenbaugh, Nathan C.; Anderson, Charles T.; Hancock, William O.

    2014-01-01

    The constituents of large, multisubunit protein complexes dictate their functions in cells, but determining their precise molecular makeup in vivo is challenging. One example of such a complex is the cellulose synthesis complex (CSC), which in plants synthesizes cellulose, the most abundant biopolymer on Earth. In growing plant cells, CSCs exist in the plasma membrane as six-lobed rosettes that contain at least three different cellulose synthase (CESA) isoforms, but the number and stoichiometry of CESAs in each CSC are unknown. To begin to address this question, we performed quantitative photobleaching of GFP-tagged AtCESA3-containing particles in living Arabidopsis thaliana cells using variable-angle epifluorescence microscopy and developed a set of information-based step detection procedures to estimate the number of GFP molecules in each particle. The step detection algorithms account for changes in signal variance due to changing numbers of fluorophores, and the subsequent analysis avoids common problems associated with fitting multiple Gaussian functions to binned histogram data. The analysis indicates that at least 10 GFP-AtCESA3 molecules can exist in each particle. These procedures can be applied to photobleaching data for any protein complex with large numbers of fluorescently tagged subunits, providing a new analytical tool with which to probe complex composition and stoichiometry. PMID:25232006

  4. Protein adsorption induced bridging flocculation: the dominant entropic pathway for nano-bio complexation

    NASA Astrophysics Data System (ADS)

    Eren, Necla Mine; Narsimhan, Ganesan; Campanella, Osvaldo H.

    2016-02-01

    Lysozyme-silica interactions and the resulting complexation were investigated through adsorption isotherms, dynamic and electrophoretic light scattering, circular dichroism (CD), and isothermal titration calorimetry (ITC). A thermodynamic analysis of ITC data revealed the existence of two binding modes during protein-nanoparticle complexation. Both binding modes are driven by the cooperation of a favorable enthalpy in the presence of a dominating entropy gain. The first binding mode has a higher binding affinity, a lower equilibrium stoichiometry and is driven by a higher entropic contribution compared to the second type. The observed favorable enthalpy gain in both modes is attributed to non-covalent complexation whereas the entropy gain is associated with the re-organization of the silica surface including not only the solvent and counter ion release, but also the protein's conformational changes. Possible mechanisms are proposed to explain non-covalent complexations for each binding mode by relating the changes in the zeta potential and hydrodynamic radius to the obtained adsorption isotherms and calorimetry profile. Based on all these findings, it is proposed that lysozyme adsorption on nano-silica is the result of protein-nanoparticle and protein-protein interactions that further leads to spontaneous, non-directional and random complexation of silica through bridging flocculation.Lysozyme-silica interactions and the resulting complexation were investigated through adsorption isotherms, dynamic and electrophoretic light scattering, circular dichroism (CD), and isothermal titration calorimetry (ITC). A thermodynamic analysis of ITC data revealed the existence of two binding modes during protein-nanoparticle complexation. Both binding modes are driven by the cooperation of a favorable enthalpy in the presence of a dominating entropy gain. The first binding mode has a higher binding affinity, a lower equilibrium stoichiometry and is driven by a higher entropic

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

    PubMed

    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.

  6. Protein Targeting and Transport as a Necessary Consequence of Increased Cellular Complexity

    PubMed Central

    Sommer, Maik S.; Schleiff, Enrico

    2014-01-01

    With increasing intracellular complexity, a new cell-biological problem that is the allocation of cytoplasmically synthesized proteins to their final destinations within the cell emerged. A special challenge is thereby the translocation of proteins into or across cellular membranes. The underlying mechanisms are only in parts well understood, but it can be assumed that the course of cellular evolution had a deep impact on the design of the required molecular machines. In this article, we aim to summarize the current knowledge and concepts of the evolutionary development of protein trafficking as a necessary premise and consequence of increased cellular complexity. PMID:25085907

  7. Quantum walks on complex networks with connection instabilities and community structure

    SciTech Connect

    Tsomokos, Dimitris I.

    2011-05-15

    A continuous-time quantum walk is investigated on complex networks with the characteristic property of community structure, which is shared by most real-world networks. Motivated by the prospect of viable quantum networks, I focus on the effects of network instabilities in the form of broken links, and examine the response of the quantum walk to such failures. It is shown that the reconfiguration of the quantum walk is determined by the community structure of the network. In this context, quantum walks based on the adjacency and Laplacian matrices of the network are compared, and their responses to link failures is analyzed.

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

    SciTech Connect

    Rutz, Natalja; Heilbronn, Regine; Weger, Stefan

    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.

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

    PubMed

    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. Graphical Abstract ᅟ.

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

    NASA Astrophysics Data System (ADS)

    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.

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

    PubMed

    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/.

  12. Protein complexed with chondroitin sulfate in poly(lactide-co-glycolide) microspheres.

    PubMed

    Lee, Eun Seong; Park, Keun-Hong; Kang, Dongmin; Park, In Suh; Min, Hyo Young; Lee, Don Haeng; Kim, Sungwon; Kim, Jong Ho; Na, Kun

    2007-06-01

    Chondroitin sulfate (CsA) is an acidic mucopolysaccharide, which is able to form ionic complexes with positively charged proteins. In this study, a protein-CsA complex was constructed to nano-sized particles. Zeta potential measurements revealed that a CsA-to-protein fraction of greater than 0.1 results in a neutralization of the positive charge on lysozyme (Lys). Based on this preliminary study, we have prepared poly(lactide-co-glycolide) (PLGA) microspheres harboring Lys/CsA complexes via the multi-emulsion method. Protein stability in the PLGA microspheres was preserved during both microsphere preparation and protein release. The profiles of Lys release from the PLGA microspheres evidenced nearly zero-order kinetics, depending on the quantity of CsA. An in vivo fluorescent image of experimental mouse tissue showed that the PLGA microspheres with the Lys/CsA complex had released the entirety of their Lys without no residual amount after 23 days, but microspheres without the complex harbored a great deal of residual Lys, which is attributable to its degradation by acidic PLGA degradates. The tissue reaction evidenced by the PLGA microspheres stabilized with CsA showed minimal foreign body reaction and little configuration of immune cells including neutrophils and macrophages, but the reactions of the PLGA microspheres without CsA were characterized by a relatively elevated inflammation. These results show that CsA is a viable candidate for long-acting micro-particular protein delivery. PMID:17337049

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

    NASA Astrophysics Data System (ADS)

    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

  14. Macromolecular composition dictates receptor and G protein selectivity of regulator of G protein signaling (RGS) 7 and 9-2 protein complexes in living cells.

    PubMed

    Masuho, Ikuo; Xie, Keqiang; Martemyanov, Kirill A

    2013-08-30

    Regulator of G protein signaling (RGS) proteins play essential roles in the regulation of signaling via G protein-coupled receptors (GPCRs). With hundreds of GPCRs and dozens of G proteins, it is important to understand how RGS regulates selective GPCR-G protein signaling. In neurons of the striatum, two RGS proteins, RGS7 and RGS9-2, regulate signaling by μ-opioid receptor (MOR) and dopamine D2 receptor (D2R) and are implicated in drug addiction, movement disorders, and nociception. Both proteins form trimeric complexes with the atypical G protein β subunit Gβ5 and a membrane anchor, R7BP. In this study, we examined GTPase-accelerating protein (GAP) activity as well as Gα and GPCR selectivity of RGS7 and RGS9-2 complexes in live cells using a bioluminescence resonance energy transfer-based assay that monitors dissociation of G protein subunits. We showed that RGS9-2/Gβ5 regulated both Gi and Go with a bias toward Go, but RGS7/Gβ5 could serve as a GAP only for Go. Interestingly, R7BP enhanced GAP activity of RGS7 and RGS9-2 toward Go and Gi and enabled RGS7 to regulate Gi signaling. Neither RGS7 nor RGS9-2 had any activity toward Gz, Gs, or Gq in the absence or presence of R7BP. We also observed no effect of GPCRs (MOR and D2R) on the G protein bias of R7 RGS proteins. However, the GAP activity of RGS9-2 showed a strong receptor preference for D2R over MOR. Finally, RGS7 displayed an four times greater GAP activity relative to RGS9-2. These findings illustrate the principles involved in establishing G protein and GPCR selectivity of striatal RGS proteins.

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

    NASA Astrophysics Data System (ADS)

    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.

  16. Mammalian actin-related protein 2/3 complex localizes to regions of lamellipodial protrusion and is composed of evolutionarily conserved proteins.

    PubMed Central

    Machesky, L M; Reeves, E; Wientjes, F; Mattheyse, F J; Grogan, A; Totty, N F; Burlingame, A L; Hsuan, J J; Segal, A W

    1997-01-01

    Human neutrophils contain a complex of proteins similar to the actin-related protein 2/3 (Arp2/3) complex of Acanthamoeba. We have obtained peptide sequence information for each member of the putative seven-protein complex previously described for Acanthamoeba and human platelets. From the peptide sequences we have identified cDNA species encoding three novel proteins in this complex. We find that in addition to Arp2 and Arp3, this complex contains a relative of the human (Suppressor of Profilin) SOP2Hs protein and four previously unknown proteins. These proteins localize in the cytoplasm of fibroblasts that lack lamellipodia, but are enriched in lamellipodia on stimulation with serum or platelet-derived growth factor. We propose a conserved and dynamic role for this complex in the organization of the actin cytoskeleton. PMID:9359840

  17. CONNECTING ANGULAR MOMENTUM AND GALACTIC DYNAMICS: THE COMPLEX INTERPLAY BETWEEN SPIN, MASS, AND MORPHOLOGY

    SciTech Connect

    Teklu, Adelheid F.; Remus, Rhea-Silvia; Dolag, Klaus; Beck, Alexander M.; Burkert, Andreas; Schulze, Felix; Steinborn, Lisa K.; Schmidt, Andreas S.

    2015-10-10

    The evolution and distribution of the angular momentum of dark matter (DM) halos have been discussed in several studies over the past decades. In particular, the idea arose that angular momentum conservation should allow us to infer the total angular momentum of the entire DM halo from measuring the angular momentum of the baryonic component, which is populating the center of the halo, especially for disk galaxies. To test this idea and to understand the connection between the angular momentum of the DM halo and its galaxy, we use a state-of-the-art, hydrodynamical cosmological simulation taken from the set of Magneticum Pathfinder simulations. Thanks to the inclusion of the relevant physical processes, the improved underlying numerical methods, and high spatial resolution, we successfully produce populations of spheroidal and disk galaxies self-consistently. Thus, we are able to study the dependence of galactic properties on their morphology. We find that (1) the specific angular momentum of stars in disk and spheroidal galaxies as a function of their stellar mass compares well with observational results; (2) the specific angular momentum of the stars in disk galaxies is slightly smaller compared to the specific angular momentum of the cold gas, in good agreement with observations; (3) simulations including the baryonic component show a dichotomy in the specific stellar angular momentum distribution when splitting the galaxies according to their morphological type (this dichotomy can also be seen in the spin parameter, where disk galaxies populate halos with slightly larger spin compared to spheroidal galaxies); (4) disk galaxies preferentially populate halos in which the angular momentum vector of the DM component in the central part shows a better alignment to the angular momentum vector of the entire halo; and (5) the specific angular momentum of the cold gas in disk galaxies is approximately 40% smaller than the specific angular momentum of the total DM halo

  18. Connecting Angular Momentum and Galactic Dynamics: The Complex Interplay between Spin, Mass, and Morphology

    NASA Astrophysics Data System (ADS)

    Teklu, Adelheid F.; Remus, Rhea-Silvia; Dolag, Klaus; Beck, Alexander M.; Burkert, Andreas; Schmidt, Andreas S.; Schulze, Felix; Steinborn, Lisa K.

    2015-10-01

    The evolution and distribution of the angular momentum of dark matter (DM) halos have been discussed in several studies over the past decades. In particular, the idea arose that angular momentum conservation should allow us to infer the total angular momentum of the entire DM halo from measuring the angular momentum of the baryonic component, which is populating the center of the halo, especially for disk galaxies. To test this idea and to understand the connection between the angular momentum of the DM halo and its galaxy, we use a state-of-the-art, hydrodynamical cosmological simulation taken from the set of Magneticum Pathfinder simulations. Thanks to the inclusion of the relevant physical processes, the improved underlying numerical methods, and high spatial resolution, we successfully produce populations of spheroidal and disk galaxies self-consistently. Thus, we are able to study the dependence of galactic properties on their morphology. We find that (1) the specific angular momentum of stars in disk and spheroidal galaxies as a function of their stellar mass compares well with observational results; (2) the specific angular momentum of the stars in disk galaxies is slightly smaller compared to the specific angular momentum of the cold gas, in good agreement with observations; (3) simulations including the baryonic component show a dichotomy in the specific stellar angular momentum distribution when splitting the galaxies according to their morphological type (this dichotomy can also be seen in the spin parameter, where disk galaxies populate halos with slightly larger spin compared to spheroidal galaxies); (4) disk galaxies preferentially populate halos in which the angular momentum vector of the DM component in the central part shows a better alignment to the angular momentum vector of the entire halo; and (5) the specific angular momentum of the cold gas in disk galaxies is approximately 40% smaller than the specific angular momentum of the total DM halo

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

    PubMed

    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

  20. A complex standard for protein identification, designed by evolution.

    PubMed

    Vaudel, Marc; Burkhart, Julia M; Breiter, Daniela; Zahedi, René P; Sickmann, Albert; Martens, Lennart

    2012-10-01

    Shotgun proteomic investigations rely on the algorithmic assignment of mass spectra to peptides. The quality of these matches is therefore a cornerstone in the analysis and has been the subject of numerous recent developments. In order to establish the benefits of novel algorithms, they are applied to reference samples of known content. However, these were recently shown to be either too simple to resemble typical real-life samples or as leading to results of lower accuracy as the method itself. Here, we describe how to use the proteome of Pyrococcus furiosus , a hyperthermophile, as a standard to evaluate proteomics identification workflows. Indeed, we prove that the Pyrococcus furiosus proteome provides a valid method for detecting random hits, comparable to the decoy databases currently in popular use, but we also prove that the Pyrococcus furiosus proteome goes squarely beyond the decoy approach by also providing many hundreds of highly reliable true positive hits. Searching the Pyrococcus furiosus proteome can thus be used as a unique test that provides the ability to reliably detect both false positives as well as proteome-scale true positives, allowing the rigorous testing of identification algorithms at the peptide and protein level.

  1. Connections between surface complexation and geometric models of mineral dissolution investigated for rhodochrosite

    NASA Astrophysics Data System (ADS)

    Duckworth, Owen W.; Martin, Scot T.

    2003-05-01

    Mineral dissolution rates have been rationalized in the literature by surface complexation models (SCM) and morphological and geometric models (GM), and reconciliation of these conceptually different yet separately highly successful models is an important goal. In the current work, morphological alterations of the surface are observed in real time at the microscopic level by atomic force microscopy (AFM) while dissolution rates are simultaneously measured at the macroscopic level by utilizing the AFM fluid cell as a classic flow-through reactor. Rhodochrosite dissolution is studied from pH = 2 to 11 at 298 K, and quantitative agreement is found between the dissolution rates determined from microscopic and macroscopic observations. Application of a SCM model for the interpretation of the kinetic data indicates that the surface concentration of >CO 3H regulates dissolution for pH < 7 while the surface concentration of >MnOH 2+ regulates dissolution for pH > 7. A GM model explains well the microscopic observations, from which it is apparent that dissolution occurs at steps associated with anisotropic pit expansion. On the basis of the observations, we combine the SCM and GM models to propose a step-site surface complexation model (SSCM), in which the dissolution rates are quantitatively related to the surface chemical speciation of steps. The governing SSCM equation is as follows: R = χ 1/2( k co + k ca)[>CO 3H] + χ 1/2( k mo + k ma)[>MnOH 2+ ], where R is the dissolution rate (mol m -2 s -1), 2χ 1/2 is the fraction of surface sites located at steps, [>CO 3H] and [>MnOH 2+ ] are surface concentrations (mol m -2), and k co, k ca, k mo, and k ma are the respective dissolution rate coefficients (s -1) for the >CO 3H and the >MnOH 2+ surface species on obtuse and acute steps. We find k co = 2.7 s -1, k ca = 2.1 × 10 -1 s -1, k mo = 4.1 × 10 -2 s -1, k ma = 3.7 × 10 -2 s -1, and χ 1/2 = 0.015 ± 0.005. The rate coefficients quantify the net result of complex surface

  2. Monte Carlo Modeling of Spectral Diffusion Employing Multiwell Protein Energy Landscapes: Application to Pigment-Protein Complexes Involved in Photosynthesis.

    PubMed

    Najafi, Mehdi; Zazubovich, Valter

    2015-06-25

    We are reporting development and initial applications of the light-induced and thermally induced spectral diffusion modeling software, covering nonphotochemical spectral hole burning (NPHB), hole recovery, and single-molecule spectroscopy and involving random generation of the multiwell protein energy landscapes. The model includes tunneling and activated barrier-hopping in both ground and excited states of a protein-chromophore system. Evolution of such a system is predicted by solving the system of rate equations. Using the barrier parameters from the range typical for the energy landscapes of the pigment-protein complexes involved in photosynthesis, we (a) show that realistic cooling of the sample must result in proteins quite far from thermodynamic equilibrium, (b) demonstrate hole evolution in the cases of burning, fixed-temperature recovery and thermocycling that mostly agrees with the experiment and modeling based on the NPHB master equation, and (c) explore the effects of different protein energy landscapes on the antihole shape. Introducing the multiwell energy landscapes and starting the hole burning experiments in realistic nonequilibrium conditions are not sufficient to explain all experimental observations even qualitatively. Therefore, for instance, one is required to invoke the modified NPHB mechanism where a complex interplay of several small conformational changes is poising the energy landscape of the pigment-protein system for downhill tunneling.

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

    PubMed Central

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

    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 analysis to identify the approximate site of the interaction and to design an appropriate linker length. Subsequently, fused constructs are generated and characterized using size exclusion chromatography and dynamic light scattering experiments. The structure of the chimeric protein is then solved by crystallization, and validated both in vitro and in vivo by substituting key interacting residues of the full length, unlinked proteins with alanine. This protocol offers the opportunity to study crucial and currently unattainable transient protein interactions involved in various biological processes. PMID:26985443

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

    PubMed Central

    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

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

    PubMed

    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

  6. Guanylate binding proteins directly attack Toxoplasma gondii via supramolecular complexes

    PubMed Central

    Kravets, Elisabeth; Degrandi, Daniel; Ma, Qijun; Peulen, Thomas-Otavio; Klümpers, Verena; Felekyan, Suren; Kühnemuth, Ralf; Weidtkamp-Peters, Stefanie; Seidel, Claus AM; Pfeffer, Klaus

    2016-01-01

    GBPs are essential for immunity against intracellular pathogens, especially for Toxoplasma gondii control. Here, the molecular interactions of murine GBPs (mGBP1/2/3/5/6), homo- and hetero-multimerization properties of mGBP2 and its function in parasite killing were investigated by mutational, Multiparameter Fluorescence Image Spectroscopy, and live cell microscopy methodologies. Control of T. gondii replication by mGBP2 requires GTP hydrolysis and isoprenylation thus, enabling reversible oligomerization in vesicle-like structures. mGBP2 undergoes structural transitions between monomeric, dimeric and oligomeric states visualized by quantitative FRET analysis. mGBPs reside in at least two discrete subcellular reservoirs and attack the parasitophorous vacuole membrane (PVM) as orchestrated, supramolecular complexes forming large, densely packed multimers comprising up to several thousand monomers. This dramatic mGBP enrichment results in the loss of PVM integrity, followed by a direct assault of mGBP2 upon the plasma membrane of the parasite. These discoveries provide vital dynamic and molecular perceptions into cell-autonomous immunity. DOI: http://dx.doi.org/10.7554/eLife.11479.001 PMID:26814575

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

    PubMed

    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.

  8. Ciliary membrane tubulin and associated proteins: a complex stable to Triton X-114 dissociation.

    PubMed

    Stephens, R E

    1985-12-19

    When either membranes from scallop gill cilia or reconstituted membranes from the same source are solubilized with Triton X-114 and the detergent is condensed by warming, no significant fraction of any major membrane protein partitions into the micellar detergent. Rather, most of the membrane lipids condense with the detergent phase, forming mixed micelles from which nearly pure lipid vesicles may be produced by adsorption of detergent with polystyrene beads. One minor membrane protein, with a molecular weight of about 20 000, is associated consistently with these vesicles. The aqueous phase contains a fairly homogeneous protein-Triton X-114 micelle sedimenting at 2.6 S in the analytical ultracentrifuge. Sucrose gradient velocity analysis in a detergent-free gradient indicates moderate size polydispersity but constant polypeptide composition throughout the sedimenting protein zone. Sucrose gradient equilibrium analysis (also in a detergent-free gradient) results in a protein-detergent complex banding at a density of 1.245 g/cm3. Sedimentation of the protein-detergent complex in the ultracentrifuge, followed by fixation and normal processing for electron microscopy, reveals a fine, reticular material consisting of 5-10-nm granules. These data are consistent with previous evidence that membrane tubulin and most other membrane proteins exist together as a discrete lipid-protein complex in molluscan gill ciliary membranes.

  9. Disassembly of synthetic Agrobacterium T-DNA–protein complexes via the host SCFVBF ubiquitin–ligase complex pathway

    PubMed Central

    Zaltsman, Adi; Lacroix, Benoît; Gafni, Yedidya; Citovsky, Vitaly

    2013-01-01

    One the most intriguing, yet least studied, aspects of the bacterium–host plant interaction is the role of the host ubiquitin/proteasome system (UPS) in the infection process. Increasing evidence indicates that pathogenic bacteria subvert the host UPS to facilitate infection. Although both mammalian and plant bacterial pathogens are known to use the host UPS, the first prokaryotic F-box protein, an essential component of UPS, was identified in Agrobacterium. During its infection, which culminates in genetic modification of the host cell, Agrobacterium transfers its T-DNA—as a complex (T-complex) with the bacterial VirE2 and host VIP1 proteins—into the host cell nucleus. There the T-DNA is uncoated from its protein components before undergoing integration into the host genome. It has been suggested that the host UPS mediates this uncoating process, but there is no evidence indicating that this activity can unmask the T-DNA molecule. Here we provide support for the idea that the plant UPS uncoats synthetic T-complexes via the Skp1/Cullin/F-box protein VBF pathway and exposes the T-DNA molecule to external enzymatic activity. PMID:23248273

  10. Probabilistic Prediction of Contacts in Protein-Ligand Complexes

    PubMed Central

    Hakulinen, Riku; Puranen, Santeri; Lehtonen, Jukka V.; Johnson, Mark S.; Corander, Jukka

    2012-01-01

    We introduce a statistical method for evaluating atomic level 3D interaction patterns of protein-ligand contacts. Such patterns can be used for fast separation of likely ligand and ligand binding site combinations out of all those that are geometrically possible. The practical purpose of this probabilistic method is for molecular docking and scoring, as an essential part of a scoring function. Probabilities of interaction patterns are calculated conditional on structural x-ray data and predefined chemical classification of molecular fragment types. Spatial coordinates of atoms are modeled using a Bayesian statistical framework with parametric 3D probability densities. The parameters are given distributions a priori, which provides the possibility to update the densities of model parameters with new structural data and use the parameter estimates to create a contact hierarchy. The contact preferences can be defined for any spatial area around a specified type of fragment. We compared calculated contact point hierarchies with the number of contact atoms found near the contact point in a reference set of x-ray data, and found that these were in general in a close agreement. Additionally, using substrate binding site in cathechol-O-methyltransferase and 27 small potential binder molecules, it was demonstrated that these probabilities together with auxiliary parameters separate well ligands from decoys (true positive rate 0.75, false positive rate 0). A particularly useful feature of the proposed Bayesian framework is that it also characterizes predictive uncertainty in terms of probabilities, which have an intuitive interpretation from the applied perspective. PMID:23155467

  11. Chloroplast isolation and affinity chromatography for enrichment of low-abundant proteins in complex proteomes.

    PubMed

    Bayer, Roman G; Stael, Simon; Teige, Markus

    2015-01-01

    Detailed knowledge of the proteome is crucial to advance the biological sciences. Low-abundant proteins are of particular interest to many biologists as they include, for example those proteins involved in signal transduction. Recent technological advances resulted in a tremendous increase in protein identification sensitivity by mass spectrometry (MS). However, the dynamic range in protein abundance still forms a fundamental problem that limits the detection of low-abundant proteins in complex proteomes. These proteins will typically escape detection in shotgun MS experiments due to the presence of other proteins at an abundance several-fold higher in order of magnitude. Therefore, specific enrichment strategies are required to overcome this technical limitation of MS-based protein discovery. We have searched for novel signal transduction proteins, more specifically kinases and calcium-binding proteins, and here we describe different approaches for enrichment of these low-abundant proteins from isolated chloroplasts from pea and Arabidopsis for subsequent proteomic analysis by MS. These approaches could be extended to include other signal transduction proteins and target different organelles. PMID:25820724

  12. Dissociation of a Dynamic Protein Complex Studied by All-Atom Molecular Simulations.

    PubMed

    Zhang, Liqun; Borthakur, Susmita; Buck, Matthias

    2016-02-23

    The process of protein complex dissociation remains to be understood at the atomic level of detail. Computers now allow microsecond timescale molecular-dynamics simulations, which make the visualization of such processes possible. Here, we investigated the dissociation process of the EphA2-SHIP2 SAM-SAM domain heterodimer complex using unrestrained all-atom molecular-dynamics simulations. Previous studies on this system have shown that alternate configurations are sampled, that their interconversion can be fast, and that the complex is dynamic by nature. Starting from different NMR-derived structures, mutants were designed to stabilize a subset of configurations by swapping ion pairs across the protein-protein interface. We focused on two mutants, K956D/D1235K and R957D/D1223R, with attenuated binding affinity compared with the wild-type proteins. In contrast to calculations on the wild-type complexes, the majority of simulations of these mutants showed protein dissociation within 2.4 μs. During the separation process, we observed domain rotation and pivoting as well as a translation and simultaneous rolling, typically to alternate and weaker binding interfaces. Several unsuccessful recapturing attempts occurred once the domains were moderately separated. An analysis of protein solvation suggests that the dissociation process correlates with a progressive loss of protein-protein contacts. Furthermore, an evaluation of internal protein dynamics using quasi-harmonic and order parameter analyses indicates that changes in protein internal motions are expected to contribute significantly to the thermodynamics of protein dissociation. Considering protein association as the reverse of the separation process, the initial role of charged/polar interactions is emphasized, followed by changes in protein and solvent dynamics. The trajectories show that protein separation does not follow a single distinct pathway, but suggest that the mechanism of dissociation is common in

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

    PubMed

    Rutz, Natalja; Heilbronn, Regine; Weger, Stefan

    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.

  14. Coarse-Grained Structure-Based Model for RNA-Protein Complexes Developed by Fluctuation Matching.

    PubMed

    Hori, Naoto; Takada, Shoji

    2012-09-11

    RNA and RNA-protein complexes have recently been intensively studied in experiments, but the corresponding molecular simulation work is much less abundant, primarily due to its large system size and the long time scale involved. Here, to overcome these bottlenecks, we develop a coarse-grained (CG) structure-based simulation model for RNA and RNA-protein complexes and test it for several molecular systems. The CG model for RNA contains three particles per nucleotide, each for phosphate, sugar, and a base. Focusing on RNA molecules that fold to well-defined native structures, we employed a structure-based potential, which is similar to the Go-like potential successfully used in CG modeling of proteins. In addition, we tested three means to approximate electrostatic interactions. Many parameters involved in the CG potential were determined via a multiscale method: We matched the native fluctuation of the CG model with that by all-atom simulations for 16 RNA molecules and 10 RNA-protein complexes, from which we derived a generic set of CG parameters. We show that the derived parameters can reproduce native fluctuations well for four RNA and two RNA-protein complexes. For tRNA, the native fluctuation in solution includes large-amplitude motions that reach conformations nearly corresponding to the hybrid state P/E and EF-Tu-bound state A/T seen in the complexes with ribosome. Finally, large-amplitude modes of ribosome are briefly described.

  15. Subunit Interactions and Organization of the Chlamydomonas reinhardtii Intraflagellar Transport Complex A Proteins*

    PubMed Central

    Behal, Robert H.; Miller, Mark S.; Qin, Hongmin; Lucker, Ben F.; Jones, Alexis; Cole, Douglas G.

    2012-01-01

    Chlamydomonas reinhardtii intraflagellar transport (IFT) particles can be biochemically resolved into two smaller assemblies, complexes A and B, that contain up to six and 15 protein subunits, respectively. We provide here the proteomic and immunological analyses that verify the identity of all six Chlamydomonas A proteins. Using sucrose density gradient centrifugation and antibody pulldowns, we show that all six A subunits are associated in a 16 S complex in both the cell bodies and flagella. A significant fraction of the cell body IFT43, however, exhibits a much slower sedimentation of ∼2 S and is not associated with the IFT A complex. To identify interactions between the six A proteins, we combined exhaustive yeast-based two-hybrid analysis, heterologous recombinant protein expression in Escherichia coli, and analysis of the newly identified complex A mutants, ift121 and ift122. We show that IFT121 and IFT43 interact directly and provide evidence for additional interactions between IFT121 and IFT139, IFT121 and IFT122, IFT140 and IFT122, and IFT140 and IFT144. The mutant analysis further allows us to propose that a subset of complex A proteins, IFT144/140/122, can form a stable 12 S subcomplex that we refer to as the IFT A core. Based on these results, we propose a model for the spatial arrangement of the six IFT A components. PMID:22170070

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

    PubMed

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

    2002-11-26

    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 interaction between Rad52 protein and RPA. Both RecO and UvsY proteins also possess the former property with regard to their cognate ssDNA-binding protein. Here, we report that RecO protein anneals ssDNA that is complexed with only its cognate ssDNA-binding protein, suggesting the involvement of species-specific interactions. Optimal activity for RecO protein occurs after formation of a 1:1 complex with SSB protein. RecR protein, which is known to stimulate RecO protein to facilitate SSB protein displacement by RecA protein, inhibits annealing by RecO protein, suggesting that RecR protein may regulate the choice between the DNA strand invasion versus annealing pathways. In addition, we show that UvsY protein anneals ssDNA; furthermore, ssDNA, which is complexed only with its cognate ssDNA-binding protein, is annealed in the presence of UvsY protein. These results indicate that RecO and possibly UvsY proteins are functional counterparts of Rad52 protein. Based on the conservation of these functions, we propose a modified double-strand break repair model that includes DNA annealing as an important intermediate step. PMID:12438681

  17. Protein complex detection via weighted ensemble clustering based on Bayesian nonnegative matrix factorization.

    PubMed

    Ou-Yang, Le; Dai, Dao-Qing; Zhang, Xiao-Fei

    2013-01-01

    Detecting protein complexes from protein-protein interaction (PPI) networks is a challenging task in computational biology. A vast number of computational methods have been proposed to undertake this task. However, each computational method is developed to capture one aspect of the network. The performance of different methods on the same network can differ substantially, even the same method may have different performance on networks with different topological characteristic. The clustering result of each computational method can be regarded as a feature that describes the PPI network from one aspect. It is therefore desirable to utilize these features to produce a more accurate and reliable clustering. In this paper, a novel Bayesian Nonnegative Matrix Factorization (NMF)-based weighted Ensemble Clustering algorithm (EC-BNMF) is proposed to detect protein complexes from PPI networks. We first apply different computational algorithms on a PPI network to generate some base clustering results. Then we integrate these base clustering results into an ensemble PPI network, in the form of weighted combination. Finally, we identify overlapping protein complexes from this network by employing Bayesian NMF model. When generating an ensemble PPI network, EC-BNMF can automatically optimize the values of weights such that the ensemble algorithm can deliver better results. Experimental results on four PPI networks of Saccharomyces cerevisiae well verify the effectiveness of EC-BNMF in detecting protein complexes. EC-BNMF provides an effective way to integrate different clustering results for more accurate and reliable complex detection. Furthermore, EC-BNMF has a high degree of flexibility in the choice of base clustering results. It can be coupled with existing clustering methods to identify protein complexes.

  18. Membrane protein complexes catalyze both 4- and 3-hydroxylation of cinnamic acid derivatives in monolignol biosynthesis.

    PubMed

    Chen, Hsi-Chuan; Li, Quanzi; Shuford, Christopher M; Liu, Jie; Muddiman, David C; Sederoff, Ronald R; Chiang, Vincent L

    2011-12-27

    The hydroxylation of 4- and 3-ring carbons of cinnamic acid derivatives during monolignol biosynthesis are key steps that determine the structure and properties of lignin. Individual enzymes have been thought to catalyze these reactions. In stem differentiating xylem (SDX) of Populus trichocarpa, two cinnamic acid 4-hydroxylases (PtrC4H1 and PtrC4H2) and a p-coumaroyl ester 3-hydroxylase (PtrC3H3) are the enzymes involved in these reactions. Here we present evidence that these hydroxylases interact, forming heterodimeric (PtrC4H1/C4H2, PtrC4H1/C3H3, and PtrC4H2/C3H3) and heterotrimeric (PtrC4H1/C4H2/C3H3) membrane protein complexes. Enzyme kinetics using yeast recombinant proteins demonstrated that the enzymatic efficiency (V(max)/k(m)) for any of the complexes is 70-6,500 times greater than that of the individual proteins. The highest increase in efficiency was found for the PtrC4H1/C4H2/C3H3-mediated p-coumaroyl ester 3-hydroxylation. Affinity purification-quantitative mass spectrometry, bimolecular fluorescence complementation, chemical cross-linking, and reciprocal coimmunoprecipitation provide further evidence for these multiprotein complexes. The activities of the recombinant and SDX plant proteins demonstrate two protein-complex-mediated 3-hydroxylation paths in monolignol biosynthesis in P. trichocarpa SDX; one converts p-coumaric acid to caffeic acid and the other converts p-coumaroyl shikimic acid to caffeoyl shikimic acid. Cinnamic acid 4-hydroxylation is also mediated by the same protein complexes. These results provide direct evidence for functional involvement of membrane protein complexes in monolignol biosynthesis.

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

    SciTech Connect

    Mena, Natalia P.; Bulteau, Anne Laure; Salazar, Julio; Hirsch, Etienne C.; Nunez, Marco T.

    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

  20. The solution structural ensembles of RNA kink-turn motifs and their protein complexes.

    PubMed

    Shi, Xuesong; Huang, Lin; Lilley, David M J; Harbury, Pehr B; Herschlag, Daniel

    2016-03-01

    With the growing number of crystal structures of RNA and RNA-protein complexes, a critical next step is understanding the dynamic solution behavior of these entities in terms of conformational ensembles and energy landscapes. To this end, we have used X-ray scattering interferometry (XSI) to probe the ubiquitous RNA kink-turn motif and its complexes with the canonical kink-turn binding protein L7Ae. XSI revealed that the folded kink-turn is best described as a restricted conformational ensemble. The ions present in solution alter the nature of this ensemble, and protein binding can perturb the kink-turn ensemble without collapsing it to a unique state. This study demonstrates how XSI can reveal structural and ensemble properties of RNAs and RNA-protein complexes and uncovers the behavior of an important RNA-protein motif. This type of information will be necessary to understand, predict and engineer the behavior and function of RNAs and their protein complexes. PMID:26727239

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

    PubMed Central

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

    PubMed Central

    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.

  3. The Solution Structural Ensembles of RNA Kink-turn Motifs and Their Protein Complexes

    PubMed Central

    Huang, Lin; Lilley, David M. J.

    2015-01-01

    With the growing number of crystal structures of RNA and RNA/protein complexes, a critical next step is understanding the dynamic behavior of these entities in solution in terms of conformational ensembles and energy landscapes. To this end, we have used X-ray scattering interferometry (XSI) to probe the widespread RNA kink-turn motif and its complexes with the canonical kink-turn binding protein L7Ae. XSI revealed that the folded kink-turn is best described as a restricted conformational ensemble. The ions present in solution alter the nature of this ensemble, and protein binding can perturb the kink-turn ensemble without collapsing it to a unique state. This study demonstrates how XSI can reveal structural and ensemble properties of RNAs and RNA/protein complexes in solution and uncovers the behavior of an important RNA/protein motif. This type of information will be necessary to understand, predict, and engineer the behavior and function of RNAs and their protein complexes. PMID:26727239

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

    PubMed

    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

  5. Luminescent Alkyne-Bearing Terbium(III) Complexes and Their Application to Bioorthogonal Protein Labeling.

    PubMed

    O'Malley, William I; Abdelkader, Elwy H; Aulsebrook, Margaret L; Rubbiani, Riccardo; Loh, Choy-Theng; Grace, Michael R; Spiccia, Leone; Gasser, Gilles; Otting, Gottfried; Tuck, Kellie L; Graham, Bim

    2016-02-15

    Two new bifunctional macrocyclic chelate ligands that form luminescent terbium(III) complexes featuring an alkyne group for conjugation to (bio)molecules via the Cu(I)-catalyzed "click" reaction were synthesized. Upon ligation, the complexes exhibit a significant luminescent enhancement when excited at the λ(max) of the "clicked" products. To demonstrate the utility of the complexes for luminescent labeling, they were conjugated in vitro to E. coli aspartate/glutamate-binding protein incorporating a genetically encoded p-azido-L-phenylalanine or p-(azidomethyl)-L-phenylalanine residue. The complexes may prove useful for time-gated assay applications.

  6. Identification of cytochrome P450 2C2 protein complexes in mouse liver.

    PubMed

    Li, Bin; Yau, Peter; Kemper, Byron

    2011-08-01

    Interactions of microsomal cytochromes P450 (CYPs) with other proteins in the microsomal membrane are important for their function. In addition to their redox partners, CYPs have been reported to interact with other proteins not directly involved in their enzymatic function. In this study, proteins were identified that interact with CYP2C2 in vivo in mouse liver. Flag-tagged CYP2C2 was expressed exogenously in mouse liver and was affinity purified, along with associated proteins which were identified by MS and confirmed by Western blotting. Over 20 proteins reproducibly copurified with CYP2C2. The heterogeneous sedimentation velocity of CYP2C2 and associated proteins by centrifugation in sucrose gradients and sequential immunoprecipitation analysis were consistent with multiple CYP2C2 complexes of differing composition. The abundance of CYPs and other drug metabolizing enzymes and NAD/NADP requiring enzymes associated with CYP2C2 suggest that complexes of these proteins may improve enzymatic efficiency or facilitate sequential metabolic steps. Chaperones, which may be important for maintaining CYP function, and reticulons, endoplasmic reticulum proteins that shape the morphology of the endoplasmic reticulum and are potential endoplasmic reticulum retention proteins for CYPs, were also associated with CYP2C2.

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

    PubMed

    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

  8. Complex chromatin condensation patterns and nuclear protein transitions during spermiogenesis: examples from mollusks.

    PubMed

    Chiva, M; Saperas, N; Ribes, E

    2011-12-01

    In this paper we review and analyze the chromatin condensation pattern during spermiogenesis in several species of mollusks. Previously, we had described the nuclear protein transitions during spermiogenesis in these species. The results of our study show two types of condensation pattern: simple patterns and complex patterns, with the following general characteristics: (a) When histones (always present in the early spermatid nucleus) are directly replaced by SNBP (sperm nuclear basic proteins) of the protamine type, the spermiogenic chromatin condensation pattern is simple. However, if the replacement is not direct but through intermediate proteins, the condensation pattern is complex. (b) The intermediate proteins found in mollusks are precursor molecules that are processed during spermiogenesis to the final protamine molecules. Some of these final protamines represent proteins with the highest basic amino acid content known to date, which results in the establishment of a very strong electrostatic interaction with DNA. (c) In some instances, the presence of complex patterns of chromatin condensation clearly correlates with the acquisition of specialized forms of the mature sperm nuclei. In contrast, simple condensation patterns always lead to rounded, oval or slightly cylindrical nuclei. (d) All known cases of complex spermiogenic chromatin condensation patterns are restricted to species with specialized sperm cells (introsperm). At the time of writing, we do not know of any report on complex condensation pattern in species with external fertilization and, therefore, with sperm cells of the primitive type (ect-aquasperm). (e) Some of the mollusk an spermiogenic chromatin condensation patterns of the complex type are very similar (almost identical) to those present in other groups of animals. Interestingly, the intermediate proteins involved in these cases can be very different.In this study, we discuss the biological significance of all these features and

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

    PubMed

    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.

  10. Alternative Eukaryotic Expression Systems for the Production of Proteins and Protein Complexes.

    PubMed

    Gómez, Sara; López-Estepa, Miguel; Fernández, Francisco J; Suárez, Teresa; Vega, M Cristina

    2016-01-01

    Besides the most established expression hosts, several eukaryotic microorganisms and filamentous fungi have also been successfully used as platforms for the production of foreign proteins. Filamentous fungi and Dictyostelium discoideum are two prominent examples. Filamentous fungi, typically Aspergillus and Trichoderma, are usually employed for the industrial production of enzymes and secondary metabolites for food processing, pharmaceutical drugs production, and textile and paper applications, with multiple products already accepted for their commercialization. The low cost of culture medium components, high secretion capability directly to the extracellular medium, and the intrinsic ability to produce post-translational modifications similar to the mammalian type, have promoted this group as successful hosts for the expression of proteins, including examples from phylogenetically distant groups: humans proteins such as IL-2, IL-6 or epithelial growth factor; α-galactosidase from plants; or endoglucanase from Cellulomonas fimi, among others. D. discoideum is a social amoeba that can be used as an expression platform for a variety of proteins, which has been extensively illustrated for cytoskeletal proteins. New vectors for heterologous expression in D. discoideum have been recently developed that might increase the usefulness of this system and expand the range of protein classes that can be tackled. Continuous developments are ongoing to improve strains, promoters, production and downstream processes for filamentous fungi, D. discoideum, and other alternative eukaryotic hosts. Either for the overexpression of individual genes, or in the coexpression of multiples genes, this chapter illustrates the enormous possibilities offered by these groups of eukaryotic organisms. PMID:27165325

  11. Alternative Eukaryotic Expression Systems for the Production of Proteins and Protein Complexes.

    PubMed

    Gómez, Sara; López-Estepa, Miguel; Fernández, Francisco J; Suárez, Teresa; Vega, M Cristina

    2016-01-01

    Besides the most established expression hosts, several eukaryotic microorganisms and filamentous fungi have also been successfully used as platforms for the production of foreign proteins. Filamentous fungi and Dictyostelium discoideum are two prominent examples. Filamentous fungi, typically Aspergillus and Trichoderma, are usually employed for the industrial production of enzymes and secondary metabolites for food processing, pharmaceutical drugs production, and textile and paper applications, with multiple products already accepted for their commercialization. The low cost of culture medium components, high secretion capability directly to the extracellular medium, and the intrinsic ability to produce post-translational modifications similar to the mammalian type, have promoted this group as successful hosts for the expression of proteins, including examples from phylogenetically distant groups: humans proteins such as IL-2, IL-6 or epithelial growth factor; α-galactosidase from plants; or endoglucanase from Cellulomonas fimi, among others. D. discoideum is a social amoeba that can be used as an expression platform for a variety of proteins, which has been extensively illustrated for cytoskeletal proteins. New vectors for heterologous expression in D. discoideum have been recently developed that might increase the usefulness of this system and expand the range of protein classes that can be tackled. Continuous developments are ongoing to improve strains, promoters, production and downstream processes for filamentous fungi, D. discoideum, and other alternative eukaryotic hosts. Either for the overexpression of individual genes, or in the coexpression of multiples genes, this chapter illustrates the enormous possibilities offered by these groups of eukaryotic organisms.

  12. Gaussian weighted neighborhood connectivity of nonlinear line attractor for learning complex manifolds

    NASA Astrophysics Data System (ADS)

    Aspiras, Theus H.; Asari, Vijayan K.; Sakla, Wesam

    2015-03-01

    The human brain has the capability to process high quantities of data quickly for detection and recognition tasks. These tasks are made simpler by the understanding of data, which intentionally removes redundancies found in higher dimensional data and maps the data onto a lower dimensional space. The brain then encodes manifolds created in these spaces, which reveal a specific state of the system. We propose to use a recurrent neural network, the nonlinear line attractor (NLA) network, for the encoding of these manifolds as specific states, which will draw untrained data towards one of the specific states that the NLA network has encoded. We propose a Gaussian-weighted modular architecture for reducing the computational complexity of the conventional NLA network. The proposed architecture uses a neighborhood approach for establishing the interconnectivity of neurons to obtain the manifolds. The modified NLA network has been implemented and tested on the Electro-Optic Synthetic Vehicle Model Database created by the Air Force Research Laboratory (AFRL), which contains a vast array of high resolution imagery with several different lighting conditions and camera views. It is observed that the NLA network has the capability for representing high dimensional data for the recognition of the objects of interest through its new learning strategy. A nonlinear dimensionality reduction scheme based on singular value decomposition has found to be very effective in providing a low dimensional representation of the dataset. Application of the reduced dimensional space on the modified NLA algorithm would provide fast and more accurate recognition performance for real time applications.

  13. TULP1 Missense Mutations Induces the Endoplasmic Reticulum Unfolded Protein Response Stress Complex (ER-UPR).

    PubMed

    Lobo, Glenn P; Ebke, Lindsey A; Au, Adrian; Hagstrom, Stephanie A

    2016-01-01

    Mutations in the TULP1 gene are associated with early-onset retinitis pigmentosa (RP); however, the molecular mechanisms related to the deleterious effects of TULP1 mutations remains unknown. Several studies have shown that misfolded proteins secondary to genetic mutations can accumulate within the endoplasmic reticulum (ER), causing activation of the unfolded protein response (UPR) complex followed by cellular apoptosis. We hypothesize that TULP1 mutations produce misfolded protein products that accumulate in the ER and induce cellular apoptosis via the UPR. To test our hypothesis, we first performed three in-silico analyses of TULP1 missense mutations (I459K, R420P and F491L), which predicted misfolded protein products. Subsequently, the three mutant TULP1-GFP constructs and wild-type (wt) TULP1-GFP were transiently transfected into hTERT-RPE-1 cells. Staining of cells using ER tracker followed by confocal microscopy showed wt-TULP1 localized predominantly to the cytoplasm and plasma membrane. In contrast, all three mutant TULP1 proteins revealed cytoplasmic punctate staining which co-localized with the ER. Furthermore, western blot analysis of cells expressing mutant TULP1 proteins revealed induction of downstream targets of the ER-UPR complex, including BiP/GPR-78, phosphorylated-PERK (Thr980) and CHOP. Our in-vitro analyses suggest that mutant TULP1 proteins are misfolded and accumulate within the ER leading to induction of the UPR stress response complex. PMID:26427415

  14. Interaction network containing conserved and essential protein complexes in Escherichia coli

    NASA Astrophysics Data System (ADS)

    Butland, Gareth; Peregrín-Alvarez, José Manuel; Li, Joyce; Yang, Wehong; Yang, Xiaochun; Canadien, Veronica; Starostine, Andrei; Richards, Dawn; Beattie, Bryan; Krogan, Nevan; Davey, Michael; Parkinson, John; Greenblatt, Jack; Emili, Andrew

    2005-02-01

    Proteins often function as components of multi-subunit complexes. Despite its long history as a model organism, no large-scale analysis of protein complexes in Escherichia coli has yet been reported. To this end, we have targeted DNA cassettes into the E. coli chromosome to create carboxy-terminal, affinity-tagged alleles of 1,000 open reading frames (~ 23% of the genome). A total of 857 proteins, including 198 of the most highly conserved, soluble non-ribosomal proteins essential in at least one bacterial species, were tagged successfully, whereas 648 could be purified to homogeneity and their interacting protein partners identified by mass spectrometry. An interaction network of protein complexes involved in diverse biological processes was uncovered and validated by sequential rounds of tagging and purification. This network includes many new interactions as well as interactions predicted based solely on genomic inference or limited phenotypic data. This study provides insight into the function of previously uncharacterized bacterial proteins and the overall topology of a microbial interaction network, the core components of which are broadly conserved across Prokaryota.

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

    SciTech Connect

    Wang, Chengliang; Zhang, Qiongdi; Hang, Tianrong; Tao, Yue; Ma, Xukai; Wu, Minhao; Zhang, Xuan Zang, Jianye

    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.

  16. The Histone Deacetylase Complex 1 Protein of Arabidopsis Has the Capacity to Interact with Multiple Proteins Including Histone 3-Binding Proteins and Histone 1 Variants1[OPEN

    PubMed Central

    Carr, Craig; Asensi-Fabado, Maria A.; Donald, Naomi A.; Hannah, Matthew A.; Amtmann, Anna

    2016-01-01

    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. PMID:26951436

  17. 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.

    PubMed

    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.

  18. The deadly connection between endoplasmic reticulum, Ca2+, protein synthesis, and the endoplasmic reticulum stress response in malignant glioma cells

    PubMed Central

    Johnson, Guyla G.; White, Misti C.; Wu, Jian-He; Vallejo, Matthew; Grimaldi, Maurizio

    2014-01-01

    Background The endoplasmic reticulum (ER) is involved in Ca2+ signaling and protein processing. Accumulation of unfolded proteins following ER Ca2+ depletion triggers the ER stress response (ERSR), which facilitates protein folding and removal of damaged proteins and can induce cell death. Unfolded proteins bind to chaperones, such as the glucose-regulated protein (GRP)78 and cause the release of GRP78-repressed proteins executing ERSR. Methods Several glioma cell lines and primary astrocytes were used to analyze ERSR using standard western blots, reverse transcription–PCR, viability assays, and single cell Ca2+ imaging. Results ERSR induction with thapsigargin results in a more intense ERSR associated with a larger loss of ER Ca2+, activation of ER-associated caspases (4/12) and caspase 3, and a higher rate of malignant glioma cell death than in normal glial cells. Malignant glioma cells have higher levels of protein synthesis and expression of the translocon (a component of the ribosomal complex, guiding protein entry in the ER), the activity of which is associated with the loss of ER Ca2+. Our experiments confirm increased expression of the translocon in malignant glioma cells. In addition, blockade of the ribosome-translocon complex with agents differently affecting translocon Ca2+ permeability causes opposite effects on ERSR deployment and death of malignant glioma cells. Conclusions Excessive ER Ca2+ loss due to translocon activity appears to be responsible for the enhancement of ERSR, leading to the death of glioma cells. The results reveal a characteristic of malignant glioma cells that could be exploited to develop new therapeutic strategies to treat incurable glial malignancies. PMID:24569545

  19. A pearl protein self-assembles to form protein complexes that amplify mineralization.

    PubMed

    Perovic, Iva; Mandal, Trinanjana; Evans, John Spencer

    2013-08-20

    The formation of the nacre pearl in marine invertebrates represents an on-demand production of mineralization in response to an irritant or parasite threat to the mantle organ. In the Japanese pearl oyster (Pinctada fucata), this process is mediated by a 12-member protein family known as PFMG (Pinctada fucata mantle gene). One of these proteins, PFGM1, has been implicated in modulating calcium carbonate crystal growth and has been reported to possess an EF-hand-like domain. In this report, we establish that the recombinant PFMG1 (rPFMG1) is an intrinsically disordered "imitator" EF-hand protein that increases the number of calcium carbonate mineral crystals that form relative to control scenarios and does not induce aragonite formation. This protein possesses a modified pseudo-EF-hand sequence at the C-terminal end which exhibits low homology (30-40%) to the pseudo-EF-hand mitochondrial SCaMCs buffering/solute transport proteins. This low sequence homology is the result of the inclusion of disorder-promoting amino acids and short amyloid-like aggregation-prone cross-β-strand sequences within the putative PFMG1 pseudo-EF-hand sequence region. Similar to other nacre proteins, rPFMG1 oligomerizes to form amorphous, heterogeneously sized protein oligomers and films in vitro, and this process is enhanced by Ca(2+), which promotes the formation of aggregation-prone extended β-strand structure within rPFMG1. From these results, we conclude that PFMG1 forms supramolecular assemblies that play an important role in amplifying the nucleation process that is crucial for coating or neutralizing invasive threats to the mantle organ. PMID:23865482

  20. A pearl protein self-assembles to form protein complexes that amplify mineralization.

    PubMed

    Perovic, Iva; Mandal, Trinanjana; Evans, John Spencer

    2013-08-20

    The formation of the nacre pearl in marine invertebrates represents an on-demand production of mineralization in response to an irritant or parasite threat to the mantle organ. In the Japanese pearl oyster (Pinctada fucata), this process is mediated by a 12-member protein family known as PFMG (Pinctada fucata mantle gene). One of these proteins, PFGM1, has been implicated in modulating calcium carbonate crystal growth and has been reported to possess an EF-hand-like domain. In this report, we establish that the recombinant PFMG1 (rPFMG1) is an intrinsically disordered "imitator" EF-hand protein that increases the number of calcium carbonate mineral crystals that form relative to control scenarios and does not induce aragonite formation. This protein possesses a modified pseudo-EF-hand sequence at the C-terminal end which exhibits low homology (30-40%) to the pseudo-EF-hand mitochondrial SCaMCs buffering/solute transport proteins. This low sequence homology is the result of the inclusion of disorder-promoting amino acids and short amyloid-like aggregation-prone cross-β-strand sequences within the putative PFMG1 pseudo-EF-hand sequence region. Similar to other nacre proteins, rPFMG1 oligomerizes to form amorphous, heterogeneously sized protein oligomers and films in vitro, and this process is enhanced by Ca(2+), which promotes the formation of aggregation-prone extended β-strand structure within rPFMG1. From these results, we conclude that PFMG1 forms supramolecular assemblies that play an important role in amplifying the nucleation process that is crucial for coating or neutralizing invasive threats to the mantle organ.

  1. Quantifying attomole amounts of proteins from complex samples by nano-LC and selected reaction monitoring.

    PubMed

    Fröhlich, Thomas; Arnold, Georg J

    2011-01-01

    Selected reaction monitoring (SRM) is one of the most powerful techniques for the relative and absolute quantification of proteins from complex protein mixtures. In contrast to traditional protein quantification methods such as ELISAs or RIAs, the SRM method uses mass spectrometry for detection. Further benefits of SRM are as follows: (1) high specificity and sensitivity; (2) large linear dynamic range of at least three orders of magnitude; and (3) the possibility to quantify multiple proteins simultaneously in a single MS run from an individual sample. To perform SRM-based protein quantification reliably, a careful design of the assay is essential, and several pitfalls must be avoided. The aim of this chapter is to help SRM newcomers to establish SRM-based protein quantification assays and discuss an overview of typical work flows that are applied during SRM assay development.

  2. Tuning cobalt(III) Schiff base complexes as activated protein inhibitors.

    PubMed

    Heffern, Marie C; Reichova, Viktorie; Coomes, Joseph L; Harney, Allison S; Bajema, Elizabeth A; Meade, Thomas J

    2015-09-21

    Cobalt(III) Schiff base complexes ([Co(acacen)(L)2](+), where L = NH3) inhibit histidine-containing proteins through dissociative exchange of the labile axial ligands (L). This work investigates axial ligand exchange dynamics of [Co(acacen)(L)2](+) complexes toward the development of protein inhibitors that are activated by external triggers such as light irradiation. We sought to investigate ligand exchange dynamics to design a Co(III) complex that is substitutionally inert under normal physiological conditions for selective activation. Fluorescent imidazoles (C3Im) were prepared as axial ligands in [Co(acacen)(L)2](+) to produce complexes (CoC3Im) that could report on ligand exchange and, thus, complex stability. These fluorescent imidazole reporters guided the design of a new dinuclear Co(III) Schiff base complex containing bridging diimidazole ligands, which exhibits enhanced stability to ligand exchange with competing imidazoles and to hydrolysis within a biologically relevant pH range. These studies inform the design of biocompatible Co(III) Schiff base complexes that can be selectively activated for protein inhibition with spatial and temporal specificity.

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

    PubMed

    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

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

    PubMed

    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.

  5. The Axial Element Protein DESYNAPTIC2 Mediates Meiotic Double-Strand Break Formation and Synaptonemal Complex Assembly in Maize.

    PubMed

    Lee, Ding Hua; Kao, Yu-Hsin; Ku, Jia-Chi; Lin, Chien-Yu; Meeley, Robert; Jan, Ya-Shiun; Wang, Chung-Ju Rachel

    2015-09-01

    During meiosis, homologous chromosomes pair and recombine via repair of programmed DNA double-strand breaks (DSBs). DSBs are formed in the context of chromatin loops, which are anchored to the proteinaceous axial element (AE). The AE later serves as a framework to assemble the synaptonemal complex (SC) that provides a transient but tight connection between homologous chromosomes. Here, we showed that DESYNAPTIC2 (DSY2), a coiled-coil protein, mediates DSB formation and is directly involved in SC assembly in maize (Zea mays). The dsy2 mutant exhibits homologous pairing defects, leading to sterility. Analyses revealed that DSB formation and the number of RADIATION SENSITIVE51 (RAD51) foci are largely reduced, and synapsis is completely abolished in dsy2 meiocytes. Super-resolution structured illumination microscopy showed that DSY2 is located on the AE and forms a distinct alternating pattern with the HORMA-domain protein ASYNAPTIC1 (ASY1). In the dsy2 mutant, localization of ASY1 is affected, and loading of the central element ZIPPER1 (ZYP1) is disrupted. Yeast two-hybrid and bimolecular fluorescence complementation experiments further demonstrated that ZYP1 interacts with DSY2 but does not interact with ASY1. Therefore, DSY2, an AE protein, not only mediates DSB formation but also bridges the AE and central element of SC during meiosis. PMID:26296964

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

    PubMed

    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.

  7. Fluorescence Anisotropy Reveals Order and Disorder of Protein Domains in the Nuclear Pore Complex

    PubMed Central

    Mattheyses, Alexa L.; Kampmann, Martin; Atkinson, Claire E.; Simon, Sanford M.

    2010-01-01

    We present a new approach for studying individual protein domains within the nuclear pore complex (NPC) using fluorescence polarization microscopy. The NPC is a large macromolecular complex, the size and complexity of which presents experimental challenges. Using fluorescence anisotropy and exploiting the symmetry of the NPC and its organization in the nuclear envelope, we have resolved order and disorder of individual protein domains. Fluorescently tagging specific domains of individual nucleoporins revealed both rigid and flexible domains: the tips of the FG domains are disordered, whereas the NPC-anchored domains are ordered. Our technique allows the collection of structural information in vivo, providing the ability to probe the organization of protein domains within the NPC. This has particular relevance for the FG domain nucleoporins, which are crucial for nucleocytoplasmic transport. PMID:20858414

  8. Protein-dependent Membrane Interaction of A Partially Disordered Protein Complex with Oleic Acid: Implications for Cancer Lipidomics

    PubMed Central

    Chaudhuri, Arunima; Prasanna, Xavier; Agiru, Priyanka; Chakraborty, Hirak; Rydström, Anna; Ho, James C. S.; Svanborg, Catharina; Sengupta, Durba; Chattopadhyay, Amitabha

    2016-01-01

    Bovine α-lactalbumin (BLA) forms cytotoxic complexes with oleic acid (OA) that perturbs tumor cell membranes, but molecular determinants of these membrane-interactions remain poorly understood. Here, we aim to obtain molecular insights into the interaction of BLA/BLA-OA complex with model membranes. We characterized the folding state of BLA-OA complex using tryptophan fluorescence and resolved residue-specific interactions of BLA with OA using molecular dynamics simulation. We integrated membrane-binding data using a voltage-sensitive probe and molecular dynamics (MD) to demonstrate the preferential interaction of the BLA-OA complex with negatively charged membranes. We identified amino acid residues of BLA and BLA-OA complex as determinants of these membrane interactions using MD, functionally corroborated by uptake of the corresponding α-LA peptides across tumor cell membranes. The results suggest that the α-LA component of these cytotoxic complexes confers specificity for tumor cell membranes through protein interactions that are maintained even in the lipid complex, in the presence of OA. PMID:27731329

  9. Protein-dependent Membrane Interaction of A Partially Disordered Protein Complex with Oleic Acid: Implications for Cancer Lipidomics

    NASA Astrophysics Data System (ADS)

    Chaudhuri, Arunima; Prasanna, Xavier; Agiru, Priyanka; Chakraborty, Hirak; Rydström, Anna; Ho, James C. S.; Svanborg, Catharina; Sengupta, Durba; Chattopadhyay, Amitabha

    2016-10-01

    Bovine α-lactalbumin (BLA) forms cytotoxic complexes with oleic acid (OA) that perturbs tumor cell membranes, but molecular determinants of these membrane-interactions remain poorly understood. Here, we aim to obtain molecular insights into the interaction of BLA/BLA-OA complex with model membranes. We characterized the folding state of BLA-OA complex using tryptophan fluorescence and resolved residue-specific interactions of BLA with OA using molecular dynamics simulation. We integrated membrane-binding data using a voltage-sensitive probe and molecular dynamics (MD) to demonstrate the preferential interaction of the BLA-OA complex with negatively charged membranes. We identified amino acid residues of BLA and BLA-OA complex as determinants of these membrane interactions using MD, functionally corroborated by uptake of the corresponding α-LA peptides across tumor cell membranes. The results suggest that the α-LA component of these cytotoxic complexes confers specificity for tumor cell membranes through protein interactions that are maintained even in the lipid complex, in the presence of OA.

  10. Membrane-mediated protein-protein interactions and connection to elastic models: a coarse-grained simulation analysis of gramicidin A association.

    PubMed

    Yoo, Jejoong; Cui, Qiang

    2013-01-01

    To further foster the connection between particle based and continuum mechanics models for membrane mediated biological processes, we carried out coarse-grained (CG) simulations of gramicidin A (gA) dimer association and analyzed the results based on the combination of potential of mean force (PMF) and stress field calculations. Similar to previous studies, we observe that the association of gA dimers depends critically on the degree of hydrophobic mismatch, with the estimated binding free energy of >10 kcal/mol in a distearoylphosphatidylcholine bilayer. Qualitative trends in the computed PMF can be understood based on the stress field distributions near a single gA dimer and between a pair of gA dimers. For example, the small PMF barrier, which is ∼1 kcal/mol independent of lipid type, can be captured nearly quantitatively by considering membrane deformation energy associated with the region confined by two gA dimers. However, the PMF well depth is reproduced poorly by a simple continuum model that only considers membrane deformation energy beyond the annular lipids. Analysis of lipid orientation, configuration entropy, and stress distribution suggests that the annular lipids make a significant contribution to the association of two gA dimers. These results highlight the importance of explicitly considering contributions from annular lipids when constructing approximate models to study processes that involve a significant reorganization of lipids near proteins, such as protein-protein association and protein insertion into biomembranes. Finally, large-scale CG simulations indicate that multiple gA dimers also form clusters, although the preferred topology depends on the protein concentration. Even at high protein concentrations, every gA dimer requires contact to lipid hydrocarbons to some degree, and at most three to four proteins are in contact with each gA dimer; this observation highlights another aspect of the importance of interactions between proteins

  11. Structure of Yeast Poly(A) Polymerase in Complex with a Peptide from Fip1, an Intrinsically Disordered Protein

    SciTech Connect

    Meinke,G.; Ezeokonkwo, C.; Balbo, P.; Stafford, W.; Moore, C.; Bohm, A.

    2008-01-01

    In yeast, the mRNA processing enzyme poly(A) polymerase is tethered to the much larger 3'-end processing complex via Fip1, a 36 kDa protein of unknown structure. We report the 2.6 Angstroms crystal structure of yeast poly(A) polymerase in complex with a peptide containing residues 80-105 of Fip1. The Fip1 peptide binds to the outside surface of the C-terminal domain of the polymerase. On the basis of this structure, we designed a mutant of the polymerase (V498Y, C485R) that is lethal to yeast. The mutant is unable to bind Fip1 but retains full polymerase activity. Fip1 is found in all eukaryotes and serves to connect poly(A) polymerase to pre-mRNA processing complexes in yeast, plants, and mammals. However, the Fip1 sequence is highly divergent, and residues on both Pap1 and Fip1 at the observed interaction surface are poorly conserved. Herein we demonstrate using analytical ultracentrifugation, circular dichroism, proteolytic studies, and other techniques that, in the absence of Pap1, Fip1 is largely, if not completely, unfolded. We speculate that flexibility may be important for Fip1's function as a molecular scaffold.

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

    PubMed

    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.

  13. Spatiotemporal Dynamics of Early DNA Damage Response Proteins on Complex DNA Lesions

    PubMed Central

    Tobias, Frank; Löb, Daniel; Lengert, Nicor; Durante, Marco; Drossel, Barbara; Taucher-Scholz, Gisela; Jakob, Burkhard

    2013-01-01

    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 repair without the

  14. Understanding the interaction of an antitumoral platinum(II) 7-azaindolate complex with proteins and DNA.

    PubMed

    Samper, Katia G; Rodríguez, Venancio; Ortega-Carrasco, Elisabeth; Atrian, Sílvia; Maréchal, Jean Didier; Cutillas, Natalia; Zamora, Ana; de Haro, Concepción; Capdevila, Mercè; Ruiz, José; Palacios, Òscar

    2014-12-01

    The reactivity of the [Pt(dmba)(aza-N1)(dmso)] complex 1, (a potential antitumoral drug with lower IC50 than cisplatin in several tumoral cell lines) with different proteins and oligonucleotides is investigated by means of mass spectrometry (ESI-TOF MS). The results obtained show a particular binding behaviour of this platinum(II) complex. The interaction of 1 with the assayed proteins apparently takes place by Pt-binding to the most accessible coordinating amino acids, presumably at the surface of the protein -this avoiding protein denaturation or degradation- with the subsequent release of one or two ligands of 1. The specific reactivity of 1 with distinct proteins allows to conclude that the substituted initial ligand (dmso or azaindolate) is indicative of the nature of the protein donor atom finally bound to the platinum(II) centre, i.e. N- or S-donor amino acid. Molecular modeling calculations suggest that the release of the azaindolate ligand is promoted by a proton transfer to the non-coordinating N present in the azaindolate ring, while the release of the dmso ligand is mainly favoured by the binding of a deprotonated Cys. The interaction of complex 1 with DNA takes always place through the release of the azaindolate ligand. Interestingly, the interaction of 1 with DNA only proceeds when the oligonucleotides are annealed forming a double strand. Complex 1 is also capable to displace ethidium bromide from DNA and it also weakly binds to DNA at the minor groove, as shown by Hoechst 33258 displacement experiments. Furthermore, complex 1 is also a good inhibitor of cathepsin B (an enzyme implicated in a number of cancer related events). Therefore, although compound 1 is definitely able to bind proteins that can hamper its arrival to the nuclear target, it should be taken into consideration as a putative anticancer drug due to its strong interaction with oligonucleotides and its effective inhibition of cat B. PMID:25106460

  15. Crystallization of the large membrane protein complex photosystem I in a microfluidic channel.

    PubMed

    Abdallah, Bahige G; Kupitz, Christopher; Fromme, Petra; Ros, Alexandra

    2013-12-23

    Traditional macroscale protein crystallization is accomplished nontrivially by exploring a range of protein concentrations and buffers in solution until a suitable combination is attained. This methodology is time-consuming and resource-intensive, hindering protein structure determination. Even more difficulties arise when crystallizing large membrane protein complexes such as photosystem I (PSI) due to their large unit cells dominated by solvent and complex characteristics that call for even stricter buffer requirements. Structure determination techniques tailored for these "difficult to crystallize" proteins such as femtosecond nanocrystallography are being developed yet still need specific crystal characteristics. Here, we demonstrate a simple and robust method to screen protein crystallization conditions at low ionic strength in a microfluidic device. This is realized in one microfluidic experiment using low sample amounts, unlike traditional methods where each solution condition is set up separately. Second harmonic generation microscopy via second-order nonlinear imaging of chiral crystals (SONICC) was applied for the detection of nanometer- and micrometer-sized PSI crystals within microchannels. To develop a crystallization phase diagram, crystals imaged with SONICC at specific channel locations were correlated to protein and salt concentrations determined by numerical simulations of the time-dependent diffusion process along the channel. Our method demonstrated that a portion of the PSI crystallization phase diagram could be reconstructed in excellent agreement with crystallization conditions determined by traditional methods. We postulate that this approach could be utilized to efficiently study and optimize crystallization conditions for a wide range of proteins that are poorly understood to date.

  16. Crystallization of the Large Membrane Protein Complex Photosystem I in a Microfluidic Channel

    PubMed Central

    Abdallah, Bahige G.; Kupitz, Christopher; Fromme, Petra; Ros, Alexandra

    2014-01-01

    Traditional macroscale protein crystallization is accomplished non-trivially by exploring a range of protein concentrations and buffers in solution until a suitable combination is attained. This methodology is time consuming and resource intensive, hindering protein structure determination. Even more difficulties arise when crystallizing large membrane protein complexes such as photosystem I (PSI) due to their large unit cells dominated by solvent and complex characteristics that call for even stricter buffer requirements. Structure determination techniques tailored for these ‘difficult to crystallize’ proteins such as femtosecond nanocrystallography are being developed, yet still need specific crystal characteristics. Here, we demonstrate a simple and robust method to screen protein crystallization conditions at low ionic strength in a microfluidic device. This is realized in one microfluidic experiment using low sample amounts, unlike traditional methods where each solution condition is set up separately. Second harmonic generation microscopy via Second Order Nonlinear Imaging of Chiral Crystals (SONICC) was applied for the detection of nanometer and micrometer sized PSI crystals within microchannels. To develop a crystallization phase diagram, crystals imaged with SONICC at specific channel locations were correlated to protein and salt concentrations determined by numerical simulations of the time-dependent diffusion process along the channel. Our method demonstrated that a portion of the PSI crystallization phase diagram could be reconstructed in excellent agreement with crystallization conditions determined by traditional methods. We postulate that this approach could be utilized to efficiently study and optimize crystallization conditions for a wide range of proteins that are poorly understood to date. PMID:24191698

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

    PubMed

    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.

    PubMed

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

    PubMed

    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.

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

    NASA Astrophysics Data System (ADS)

    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. Rational Design of Orthogonal Multipolar Interactions with Fluorine in Protein-Ligand Complexes.

    PubMed

    Pollock, Jonathan; Borkin, Dmitry; Lund, George; Purohit, Trupta; Dyguda-Kazimierowicz, Edyta; Grembecka, Jolanta; Cierpicki, Tomasz

    2015-09-24

    Multipolar interactions involving fluorine and the protein backbone have been frequently observed in protein-ligand complexes. Such fluorine-backbone interactions may substantially contribute to the high affinity of small molecule inhibitors. Here we found that introduction of trifluoromethyl groups into two different sites in the thienopyrimidine class of menin-MLL inhibitors considerably improved their inhibitory activity. In both cases, trifluoromethyl groups are engaged in short interactions with the backbone of menin. In order to understand the effect of fluorine, we synthesized a series of analogues by systematically changing the number of fluorine atoms, and we determined high-resolution crystal structures of the complexes with menin. We found that introduction of fluorine at favorable geometry for interactions with backbone carbonyls may improve the activity of menin-MLL inhibitors as much as 5- to 10-fold. In order to facilitate the design of multipolar fluorine-backbone interactions in protein-ligand complexes, we developed a computational algorithm named FMAP, which calculates fluorophilic sites in proximity to the protein backbone. We demonstrated that FMAP could be used to rationalize improvement in the activity of known protein inhibitors upon introduction of fluorine. Furthermore, FMAP may also represent a valuable tool for designing new fluorine substitutions and support ligand optimization in drug discovery projects. Analysis of the menin-MLL inhibitor complexes revealed that the backbone in secondary structures is particularly accessible to the interactions with fluorine. Considering that secondary structure elements are frequently exposed at protein interfaces, we postulate that multipolar fluorine-backbone interactions may represent a particularly attractive approach to improve inhibitors of protein-protein interactions.

  2. High Throughput Identification, Purification and Structural Characterization of Water Soluble Protein Complexes in Desulfovibrio vulgaris

    SciTech Connect

    Dong,, Ming; Han, Bong-Gyoon; Liu, Hui-Hai; Malik, J.; Geller, Jil; Yang, Li; Choi, M.; Chandonia, John-Marc; Arbelaez, Pablo; Sterling, H. J.; Typke, Dieter; Shatsky, Max; Brenner, Steve; Fisher, Susan; Williams, Evan; Szakal, Evelin; Allen, S.; Hall, S. C.; Hazen, Terry; Witkowska, H. E.; Jin, Jiming; Glaeser, Robert; Biggin, Mark

    2010-05-17

    Our scheme for the tagless purification of water soluble complexes. 10 g of protein from a crude bacterial extract is first fractionated by ammonium sulfate precipitation and then by a series of chromatographic steps: anion exchange (IEX), hydrophobic interaction (HIC), and finally size exclusion (Gel Filtration). Fractions from the last chromatography step are trypsin digested and peptides labeled with iTRAQ reagents to allow multiplexing and quantitation during mass spectrometric analysis. Elution profiles of identified proteins are then subjected to clustering analysis.

  3. The 5-S RNA . protein complex from an extreme halophile, Halobacterium cutirubrum. Purification and characterization.

    PubMed

    Smith, N; Matheson, A T; Yaguchi, M; Willick, G E; Nazar, R N

    1978-09-01

    A 5-S RNA . protein complex has been isolated from the 50-S ribosomal subunit of an extreme halophile, Halobacterium cutirubrum. The 50-S ribosomal subunit from the extreme halophile requires 3.4 M K+ and 100 mM Mg2+ for stability. However, if the high K+ concentration is maintained but the Mg2+ concentration lowered to 0.3 mM, the 5-S RNA . protein complex is selectively extracted from the subunit. After being purified on an Agarose 0.5-m column the complex had a molecular weight of about 80000 and contained 5-S RNA and two proteins, HL13 and HL19, with molecular weights (by sedimentation equilibrium) of 18700 and 18000, respectively. No ATPase or GTPase activity could be detected in the 5-S RNA . protein complex. The amino acid composition and electrophoretic mobility on polyacrylamide gels indicated both proteins were much more acidic than the equivalent from Escherichia coli or Bacillus stearothermophilus. Partial amino acid sequence data suggest HL13 is homologous to EL18 and HL19 to EL5.

  4. Isolation of a novel lutein-protein complex from Chlorella vulgaris and its functional properties.

    PubMed

    Cai, Xixi; Huang, Qimin; Wang, Shaoyun

    2015-06-01

    A novel kind of lutein-protein complex (LPC) was extracted from heterotrophic Chlorella vulgaris through aqueous extraction. The purification procedure contained solubilization of thylakoid proteins by a zwitterionic detergent CHAPS, anion exchange chromatography and gel filtration chromatography. Both wavelength scanning and HPLC analysis confirmed that lutein was the major pigment of the protein-based complex, and the mass ratio of lutein and protein was determined to be 9.72 : 100. Besides showing lipid peroxidation inhibition activity in vitro, LPC exerted significant antioxidant effects against ABTS and DPPH radicals with IC50 of 2.90 and 97. 23 μg mL(-1), respectively. Meanwhile, in vivo antioxidant activity of the complex was evaluated using the mice hepatotoxicity model; LPC significantly suppressed the carbon tetrachloride-induced elevation of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities, and decreased hepatic malondialdehyde (MDA) levels and the hepatosomatic index. Moreover, LPC could effectively restore the activities of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px) in the treated mice livers. Our findings further the progress in the research of natural protein-based lutein complexes, suggesting that LPC has the potential in hepatoprotection against chemical induced toxicity and in increasing the antioxidant capacity of the defense system in the human body.

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

    NASA Astrophysics Data System (ADS)

    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.

  6. The CRP/MLP/TLP family of LIM domain proteins: acting by connecting.

    PubMed

    Weiskirchen, Ralf; Günther, Kalle

    2003-02-01

    In vertebrates, members of the cysteine-rich protein (CRP) family are characterized by the presence of two LIM domains linked to short glycine-rich repeats. These proteins mediate protein-protein interactions and are of fundamental importance for cell differentiation, cytoskeletal remodeling, and transcriptional regulation. To date, a vast amount of information about vertebrate CRPs has become available, including their biological functions, interacting partners, and three-dimensional structures. Compatible with a molecular adapter ro