Science.gov

Sample records for protein complex connectivity

  1. Protein Connectivity in Chemotaxis Receptor Complexes.

    PubMed

    Eismann, Stephan; Endres, Robert G

    2015-12-01

    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. 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

  3. Characterization of known protein complexes using k-connectivity and other topological measures

    PubMed Central

    Gallagher, Suzanne R; Goldberg, Debra S

    2015-01-01

    Many protein complexes are densely packed, so proteins within complexes often interact with several other proteins in the complex. Steric constraints prevent most proteins from simultaneously binding more than a handful of other proteins, regardless of the number of proteins in the complex. Because of this, as complex size increases, several measures of the complex decrease within protein-protein interaction networks. However, k-connectivity, the number of vertices or edges that need to be removed in order to disconnect a graph, may be consistently high for protein complexes. The property of k-connectivity has been little used previously in the investigation of protein-protein interactions. To understand the discriminative power of k-connectivity and other topological measures for identifying unknown protein complexes, we characterized these properties in known Saccharomyces cerevisiae protein complexes in networks generated both from highly accurate X-ray crystallography experiments which give an accurate model of each complex, and also as the complexes appear in high-throughput yeast 2-hybrid studies in which new complexes may be discovered. We also computed these properties for appropriate random subgraphs.We found that clustering coefficient, mutual clustering coefficient, and k-connectivity are better indicators of known protein complexes than edge density, degree, or betweenness. This suggests new directions for future protein complex-finding algorithms. PMID:26913183

  4. A novel protein complex identification algorithm based on Connected Affinity Clique Extension (CACE).

    PubMed

    Li, Peng; He, Tingting; Hu, Xiaohua; Zhao, Junmin; Shen, Xianjun; Zhang, Ming; Wang, Yan

    2014-06-01

    A novel algorithm based on Connected Affinity Clique Extension (CACE) for mining overlapping functional modules in protein interaction network is proposed in this paper. In this approach, the value of protein connected affinity which is inferred from protein complexes is interpreted as the reliability and possibility of interaction. The protein interaction network is constructed as a weighted graph, and the weight is dependent on the connected affinity coefficient. The experimental results of our CACE in two test data sets show that the CACE can detect the functional modules much more effectively and accurately when compared with other state-of-art algorithms CPM and IPC-MCE. PMID:24803142

  5. Mining Temporal Protein Complex Based on the Dynamic PIN Weighted with Connected Affinity and Gene Co-Expression

    PubMed Central

    Shen, Xianjun; Jiang, Xingpeng; He, Tingting; Hu, Xiaohua; Yang, Jincai

    2016-01-01

    The identification of temporal protein complexes would make great contribution to our knowledge of the dynamic organization characteristics in protein interaction networks (PINs). Recent studies have focused on integrating gene expression data into static PIN to construct dynamic PIN which reveals the dynamic evolutionary procedure of protein interactions, but they fail in practice for recognizing the active time points of proteins with low or high expression levels. We construct a Time-Evolving PIN (TEPIN) with a novel method called Deviation Degree, which is designed to identify the active time points of proteins based on the deviation degree of their own expression values. Owing to the differences between protein interactions, moreover, we weight TEPIN with connected affinity and gene co-expression to quantify the degree of these interactions. To validate the efficiencies of our methods, ClusterONE, CAMSE and MCL algorithms are applied on the TEPIN, DPIN (a dynamic PIN constructed with state-of-the-art three-sigma method) and SPIN (the original static PIN) to detect temporal protein complexes. Each algorithm on our TEPIN outperforms that on other networks in terms of match degree, sensitivity, specificity, F-measure and function enrichment etc. In conclusion, our Deviation Degree method successfully eliminates the disadvantages which exist in the previous state-of-the-art dynamic PIN construction methods. Moreover, the biological nature of protein interactions can be well described in our weighted network. Weighted TEPIN is a useful approach for detecting temporal protein complexes and revealing the dynamic protein assembly process for cellular organization. PMID:27100396

  6. 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

  7. 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.

  8. 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

  9. 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…

  10. Reconfiguring the connectivity of a multiprotein complex: fusions of yeast TATA-binding protein with Brf1, and the function of transcription factor IIIB.

    PubMed

    Kassavetis, George A; Soragni, Elisabetta; Driscoll, Robert; Geiduschek, E Peter

    2005-10-25

    Transcription factor (TF) IIIB, the central transcription initiation factor of RNA polymerase III (pol III), is composed of three subunits, Bdp1, Brf1 and TATA-binding protein (TBP), all essential for normal function in vivo and in vitro. Brf1 is a modular protein: Its N-proximal half is related to TFIIB and binds similarly to the C-terminal stirrup of TBP; its C-proximal one-third provides most of the affinity for TBP by binding along the entire length of the convex surface and N-terminal lateral face of TBP. A structure-informed triple fusion protein, with TBP core placed between the N- and C-proximal domains of Brf1, has been constructed. The Brf1-TBP triple fusion protein effectively replaces both Brf1 and TBP in TFIIIC-dependent and -independent transcription in vitro, and forms extremely stable TFIIIB-DNA complexes that are indistinguishable from wild-type TFIIIB-DNA complexes by chemical nuclease footprinting. Unlike Brf1 and TBP, the triple fusion protein is able to recruit pol III for TATA box-directed transcription of linear and supercoiled DNA in the absence of Bdp1. The Brf1-TBP triple fusion protein also effectively replaces Brf1 function in vivo as the intact protein, creating a TBP paralogue in yeast that is privatized for pol III transcription. PMID:16227432

  11. Temporal connectivity in a prairie pothole complex

    USGS Publications Warehouse

    Leibowitz, S.G.; Vining, K.C.

    2003-01-01

    A number of studies have noted the occurrence of intermittent surface-water connections between depressional wetlands in general and prairie potholes in particular. Yet, the ecological implications of such connections remain largely unexplored. In 1995, we observed spillage into and out of a North Dakota wetland during two field visits. Between May 3 and May 26, there was a positive relationship between specific conductance and water level at this site, suggesting an external source of dissolved ions. We estimated that specific conductance may have increased at the site by as much as 614 ??S cm-1 due to spillage from the upslope wetland. Based on a spatial analysis that compared National Wetlands Inventory maps with 1996 color infrared imagery, we estimated that 28% of the area's wetlands had a temporary surface water connection to at least one other wetland at that time, including one complex of 14 interconnected wetlands. These results indicate that the connectivity observed in 1995 was not confined to the two wetlands nor to that single year. The degree of connectivity we observed would be expected to occur during the wetter portions of the region's 20-year wet-dry cycle. We hypothesize that intermittent surface-water connections between wetlands occur throughout the prairie pothole region. Given patterns in relief and precipitation, these connections most likely would have occurred in the eastern portion of the prairie pothole region. However, wetland drainage may have altered historical patterns. The implication of these spatial and temporal trends is that surface-water connections between depressional wetlands should be viewed as a probability event that has some distribution over time and space. We refer to connections that are impermanent, temporally discontinuous, or sporadic as temporal connectivity. The most intriguing feature of these temporary connections may be that they could affect biodiversity or population dynamics through transport of individuals

  12. Length, protein protein interactions, and complexity

    NASA Astrophysics Data System (ADS)

    Tan, Taison; Frenkel, Daan; Gupta, Vishal; Deem, Michael W.

    2005-05-01

    The evolutionary reason for the increase in gene length from archaea to prokaryotes to eukaryotes observed in large-scale genome sequencing efforts has been unclear. We propose here that the increasing complexity of protein-protein interactions has driven the selection of longer proteins, as they are more able to distinguish among a larger number of distinct interactions due to their greater average surface area. Annotated protein sequences available from the SWISS-PROT database were analyzed for 13 eukaryotes, eight bacteria, and two archaea species. The number of subcellular locations to which each protein is associated is used as a measure of the number of interactions to which a protein participates. Two databases of yeast protein-protein interactions were used as another measure of the number of interactions to which each S. cerevisiae protein participates. Protein length is shown to correlate with both number of subcellular locations to which a protein is associated and number of interactions as measured by yeast two-hybrid experiments. Protein length is also shown to correlate with the probability that the protein is encoded by an essential gene. Interestingly, average protein length and number of subcellular locations are not significantly different between all human proteins and protein targets of known, marketed drugs. Increased protein length appears to be a significant mechanism by which the increasing complexity of protein-protein interaction networks is accommodated within the natural evolution of species. Consideration of protein length may be a valuable tool in drug design, one that predicts different strategies for inhibiting interactions in aberrant and normal pathways.

  13. Algorithmic complexity of a protein

    NASA Astrophysics Data System (ADS)

    Dewey, T. Gregory

    1996-07-01

    The information contained in a protein's amino acid sequence dictates its three-dimensional structure. To quantitate the transfer of information that occurs in the protein folding process, the Kolmogorov information entropy or algorithmic complexity of the protein structure is investigated. The algorithmic complexity of an object provides a means of quantitating its information content. Recent results have indicated that the algorithmic complexity of microstates of certain statistical mechanical systems can be estimated from the thermodynamic entropy. In the present work, it is shown that the algorithmic complexity of a protein is given by its configurational entropy. Using this result, a quantitative estimate of the information content of a protein's structure is made and is compared to the information content of the sequence. Additionally, the mutual information between sequence and structure is determined. It is seen that virtually all the information contained in the protein structure is shared with the sequence.

  14. 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

  15. 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.

  16. HKC: an algorithm to predict protein complexes in protein-protein interaction networks.

    PubMed

    Wang, Xiaomin; Wang, Zhengzhi; Ye, Jun

    2011-01-01

    With the availability of more and more genome-scale protein-protein interaction (PPI) networks, research interests gradually shift to Systematic Analysis on these large data sets. A key topic is to predict protein complexes in PPI networks by identifying clusters that are densely connected within themselves but sparsely connected with the rest of the network. In this paper, we present a new topology-based algorithm, HKC, to detect protein complexes in genome-scale PPI networks. HKC mainly uses the concepts of highest k-core and cohesion to predict protein complexes by identifying overlapping clusters. The experiments on two data sets and two benchmarks show that our algorithm has relatively high F-measure and exhibits better performance compared with some other methods. PMID:22174556

  17. Binding Efficiency of Protein-Protein Complexes

    PubMed Central

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

    2012-01-01

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

  18. PCDq: human protein complex database with quality index which summarizes different levels of evidences of protein complexes predicted from H-Invitational protein-protein interactions integrative dataset

    PubMed Central

    2012-01-01

    Background Proteins interact with other proteins or biomolecules in complexes to perform cellular functions. Existing protein-protein interaction (PPI) databases and protein complex databases for human proteins are not organized to provide protein complex information or facilitate the discovery of novel subunits. Data integration of PPIs focused specifically on protein complexes, subunits, and their functions. Predicted candidate complexes or subunits are also important for experimental biologists. Description Based on integrated PPI data and literature, we have developed a human protein complex database with a complex quality index (PCDq), which includes both known and predicted complexes and subunits. We integrated six PPI data (BIND, DIP, MINT, HPRD, IntAct, and GNP_Y2H), and predicted human protein complexes by finding densely connected regions in the PPI networks. They were curated with the literature so that missing proteins were complemented and some complexes were merged, resulting in 1,264 complexes comprising 9,268 proteins with 32,198 PPIs. The evidence level of each subunit was assigned as a categorical variable. This indicated whether it was a known subunit, and a specific function was inferable from sequence or network analysis. To summarize the categories of all the subunits in a complex, we devised a complex quality index (CQI) and assigned it to each complex. We examined the proportion of consistency of Gene Ontology (GO) terms among protein subunits of a complex. Next, we compared the expression profiles of the corresponding genes and found that many proteins in larger complexes tend to be expressed cooperatively at the transcript level. The proportion of duplicated genes in a complex was evaluated. Finally, we identified 78 hypothetical proteins that were annotated as subunits of 82 complexes, which included known complexes. Of these hypothetical proteins, after our prediction had been made, four were reported to be actual subunits of the

  19. LINC complex proteins in cardiac structure, function, and disease

    PubMed Central

    Stroud, Matthew J; Banerjee, Indroneal; Lowe, Jennifer; Chen, Ju

    2014-01-01

    The LINC (LInker of Nucleoskeleton and Cytoskeleton) complex, composed of proteins within the inner and the outer nuclear membranes, connects the nuclear lamina to the cytoskeleton. The importance of this complex has been highlighted by the discovery of mutations in genes encoding LINC complex proteins, which are causative for skeletal or cardiac myopathies. Herein, this review summarizes structure, function, and interactions of major components of the LINC complex, highlights how mutations in these proteins may lead to cardiac disease, and outlines future challenges in the field. PMID:24481844

  20. Interaction graph mining for protein complexes using local clique merging.

    PubMed

    Li, Xiao-Li; Tan, Soon-Heng; Foo, Chuan-Sheng; Ng, See-Kiong

    2005-01-01

    While recent technological advances have made available large datasets of experimentally-detected pairwise protein-protein interactions, there is still a lack of experimentally-determined protein complex data. To make up for this lack of protein complex data, we explore the mining of existing protein interaction graphs for protein complexes. This paper proposes a novel graph mining algorithm to detect the dense neighborhoods (highly connected regions) in an interaction graph which may correspond to protein complexes. Our algorithm first locates local cliques for each graph vertex (protein) and then merge the detected local cliques according to their affinity to form maximal dense regions. We present experimental results with yeast protein interaction data to demonstrate the effectiveness of our proposed method. Compared with other existing techniques, our predicted complexes can match or overlap significantly better with the known protein complexes in the MIPS benchmark database. Novel protein complexes were also predicted to help biologists in their search for new protein complexes. PMID:16901108

  1. GECluster: a novel protein complex prediction method

    PubMed Central

    Su, Lingtao; Liu, Guixia; Wang, Han; Tian, Yuan; Zhou, Zhihui; Han, Liang; Yan, Lun

    2014-01-01

    Identification of protein complexes is of great importance in the understanding of cellular organization and functions. Traditional computational protein complex prediction methods mainly rely on the topology of protein–protein interaction (PPI) networks but seldom take biological information of proteins (such as Gene Ontology (GO)) into consideration. Meanwhile, the environment relevant analysis of protein complex evolution has been poorly studied, partly due to the lack of high-precision protein complex datasets. In this paper, a combined PPI network is introduced to predict protein complexes which integrate both GO and expression value of relevant protein-coding genes. A novel protein complex prediction method GECluster (Gene Expression Cluster) was proposed based on a seed node expansion strategy, in which a combined PPI network was utilized. GECluster was applied to a training combined PPI network and it predicted more credible complexes than peer methods. The results indicate that using a combined PPI network can efficiently improve protein complex prediction accuracy. In order to study protein complex evolution within cells due to changes in the living environment surrounding cells, GECluster was applied to seven combined PPI networks constructed using the data of a test set including yeast response to stress throughout a wine fermentation process. Our results showed that with the rise of alcohol concentration, protein complexes within yeast cells gradually evolve from one state to another. Besides this, the number of core and attachment proteins within a protein complex both changed significantly. PMID:26019559

  2. 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.

  3. SWISS-PROT: connecting biomolecular knowledge via a protein database.

    PubMed

    Gasteiger, E; Jung, E; Bairoch, A

    2001-07-01

    With the explosive growth of biological data, the development of new means of data storage was needed. More and more often biological information is no longer published in the conventional way via a publication in a scientific journal, but only deposited into a database. In the last two decades these databases have become essential tools for researchers in biological sciences. Biological databases can be classified according to the type of information they contain. There are basically three types of sequence-related databases (nucleic acid sequences, protein sequences and protein tertiary structures) as well as various specialized data collections. It is important to provide the users of biomolecular databases with a degree of integration between these databases as by nature all of these databases are connected in a scientific sense and each one of them is an important piece to biological complexity. In this review we will highlight our effort in connecting biological information as demonstrated in the SWISS-PROT protein database. PMID:11488411

  4. 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.

  5. 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

  6. 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.

  7. Immunoisolation of Protein Complexes from Xenopus

    PubMed Central

    Conlon, Frank L.; Miteva, Yana; Kaltenbrun, Erin; Waldron, Lauren; Greco, Todd M.; Cristea, Ileana M.

    2013-01-01

    The immunoaffinity isolation of protein complexes is an essential technique for the purification and concentration of protein complexes from cells and tissues. In this chapter we present the methodologies for the purification of proteins and protein complexes from Xenopus laev is and Xenopus tropical is. Specific to this protocol are the techniques for the cryolysis of Xenopus cells and tissues, a procedure that limits contamination from yolk proteins while preserving endogenous protein complexes, the methodologies for immunoaffinity purification of proteins using magnetic beads, and the protocols for western blot analysis. In addition, the procedures in this chapter can be extended to use with proteomic analysis of protein complexes as presented in the following chapter. PMID:22956099

  8. 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

  9. 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.

  10. Trapping mammalian protein complexes in viral particles

    PubMed Central

    Eyckerman, Sven; Titeca, Kevin; Van Quickelberghe, Emmy; Cloots, Eva; Verhee, Annick; Samyn, Noortje; De Ceuninck, Leentje; Timmerman, Evy; De Sutter, Delphine; Lievens, Sam; Van Calenbergh, Serge; Gevaert, Kris; Tavernier, Jan

    2016-01-01

    Cell lysis is an inevitable step in classical mass spectrometry–based strategies to analyse protein complexes. Complementary lysis conditions, in situ cross-linking strategies and proximal labelling techniques are currently used to reduce lysis effects on the protein complex. We have developed Virotrap, a viral particle sorting approach that obviates the need for cell homogenization and preserves the protein complexes during purification. By fusing a bait protein to the HIV-1 GAG protein, we show that interaction partners become trapped within virus-like particles (VLPs) that bud from mammalian cells. Using an efficient VLP enrichment protocol, Virotrap allows the detection of known binary interactions and MS-based identification of novel protein partners as well. In addition, we show the identification of stimulus-dependent interactions and demonstrate trapping of protein partners for small molecules. Virotrap constitutes an elegant complementary approach to the arsenal of methods to study protein complexes. PMID:27122307

  11. Improved method for protein complex detection using bottleneck proteins

    PubMed Central

    2013-01-01

    Background Detecting protein complexes is one of essential and fundamental tasks in understanding various biological functions or processes. Therefore accurate identification of protein complexes is indispensable. Methods For more accurate detection of protein complexes, we propose an algorithm which detects dense protein sub-networks of which proteins share closely located bottleneck proteins. The proposed algorithm is capable of finding protein complexes which allow overlapping with each other. Results We applied our algorithm to several PPI (Protein-Protein Interaction) networks of Saccharomyces cerevisiae and Homo sapiens, and validated our results using public databases of protein complexes. The prediction accuracy was even more improved over our previous work which used also bottleneck information of the PPI network, but showed limitation when predicting small-sized protein complex detection. Conclusions Our algorithm resulted in overlapping protein complexes with significantly improved F1 score over existing algorithms. This result comes from high recall due to effective network search, as well as high precision due to proper use of bottleneck information during the network search. PMID:23566214

  12. Metabolic Adaptation and Protein Complexes in Prokaryotes

    PubMed Central

    Krüger, Beate; Liang, Chunguang; Prell, Florian; Fieselmann, Astrid; Moya, Andres; Schuster, Stefan; Völker, Uwe; Dandekar, Thomas

    2012-01-01

    Protein complexes are classified and have been charted in several large-scale screening studies in prokaryotes. These complexes are organized in a factory-like fashion to optimize protein production and metabolism. Central components are conserved between different prokaryotes; major complexes involve carbohydrate, amino acid, fatty acid and nucleotide metabolism. Metabolic adaptation changes protein complexes according to environmental conditions. Protein modification depends on specific modifying enzymes. Proteins such as trigger enzymes display condition-dependent adaptation to different functions by participating in several complexes. Several bacterial pathogens adapt rapidly to intracellular survival with concomitant changes in protein complexes in central metabolism and optimize utilization of their favorite available nutrient source. Regulation optimizes protein costs. Master regulators lead to up- and downregulation in specific subnetworks and all involved complexes. Long protein half-life and low level expression detaches protein levels from gene expression levels. However, under optimal growth conditions, metabolite fluxes through central carbohydrate pathways correlate well with gene expression. In a system-wide view, major metabolic changes lead to rapid adaptation of complexes and feedback or feedforward regulation. Finally, prokaryotic enzyme complexes are involved in crowding and substrate channeling. This depends on detailed structural interactions and is verified for specific effects by experiments and simulations. PMID:24957769

  13. Predicting Physical Interactions between Protein Complexes*

    PubMed Central

    Clancy, Trevor; Rødland, Einar Andreas; Nygard, Ståle; Hovig, Eivind

    2013-01-01

    Protein complexes enact most biochemical functions in the cell. Dynamic interactions between protein complexes are frequent in many cellular processes. As they are often of a transient nature, they may be difficult to detect using current genome-wide screens. Here, we describe a method to computationally predict physical interactions between protein complexes, applied to both humans and yeast. We integrated manually curated protein complexes and physical protein interaction networks, and we designed a statistical method to identify pairs of protein complexes where the number of protein interactions between a complex pair is due to an actual physical interaction between the complexes. An evaluation against manually curated physical complex-complex interactions in yeast revealed that 50% of these interactions could be predicted in this manner. A community network analysis of the highest scoring pairs revealed a biologically sensible organization of physical complex-complex interactions in the cell. Such analyses of proteomes may serve as a guide to the discovery of novel functional cellular relationships. PMID:23438732

  14. 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

  15. Proteins as paradigms of complex systems.

    SciTech Connect

    Fenimore, P. W.; Frauenfelder, Hans,; Young, R. D.

    2003-03-26

    The science of complexity has moved to center stage within the past few decades. Complex systems range from glasses to the immune system and the brain. Glasses are too simple to possess all aspects of complexity; brains are too complex to expose common concepts and laws of complexity. Proteins, however, are systems where many concepts and laws of complexity can be explored experimentally, theoretically, and computationally. Such studies have elucidated crucial aspects. The energy landscape has emerged as one central concept; it describes the free energy of a system as a function of temperature and the coordinates of all relevant atoms. A second concept is that of fluctuations. Without fluctuations, proteins would be dead and life impossible. A third concept is slaving. Proteins are not isolated systems; they are embedded in cells and membranes. Slaving arises when the fluctuations in the surroundings of a protein dominate many of the motions of the protein proper.

  16. Building a Hierarchical Organization of Protein Complexes Out of Protein Association Data

    PubMed Central

    Stojmirović, Aleksandar; Yu, Yi-Kuo

    2014-01-01

    Organizing experimentally determined protein associations as a hierarchy can be a good approach to elucidating the content of protein complexes and the modularity of subcomplexes. Several challenges exist. First, intrinsically sticky proteins, such as chaperones, are often falsely assigned to many functionally unrelated complexes. Second, the reported collections of proteins may not be true “complexes” in the sense that they bind together and perform a joint cellular function. Third, due to imperfect sensitivity of protein detection methods, both false positive and false negative assignments of a protein to complexes may occur. We mitigate the first issue by down-weighting sticky proteins by their occurrence frequencies. We approach the other two problems by merging nearly identical complexes and by constructing a directed acyclic graph (DAG) based on the relationship of partial inclusion. The constructed DAG, within which smaller complexes form parts of the larger, can reveal how different complexes are joined. By merging almost identical complexes one can deemphasize the influence of false positives, while allowing false negatives to be rescued by other nearly identical association data. We investigate several protein weighting schemes and compare their corresponding DAGs using yeast and human complexes. We find that the scheme incorporating weights based on information flow in the network of direct protein–protein interactions produces biologically most meaningful DAGs. In either yeast or human, isolated nodes form a large proportion of the final hierarchy. While most connected components encompass very few nodes, the largest one for each species contains a sizable portion of all nodes. By considering examples of subgraphs composed of nodes containing a specified protein, we illustrate that the graphs' topological features can correctly suggest the biological roles of protein complexes. The input data, final results and the source code are available at ftp

  17. 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

  18. 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

  19. Protein Complex Purification by Affinity Capture.

    PubMed

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

    2016-01-01

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

  20. Systematic Characterization of Human Protein Complexes Identifies Chromosome Segregation Proteins

    PubMed Central

    Hutchins, James R.A.; Toyoda, Yusuke; Hegemann, Björn; Poser, Ina; Hériché, Jean-Karim; Sykora, Martina M.; Augsburg, Martina; Hudecz, Otto; Buschhorn, Bettina A.; Bulkescher, Jutta; Conrad, Christian; Comartin, David; Schleiffer, Alexander; Sarov, Mihail; Pozniakovsky, Andrei; Slabicki, Mikolaj Michal; Schloissnig, Siegfried; Steinmacher, Ines; Leuschner, Marit; Ssykor, Andrea; Lawo, Steffen; Pelletier, Laurence; Stark, Holger; Nasmyth, Kim; Ellenberg, Jan; Durbin, Richard; Buchholz, Frank; Mechtler, Karl; Hyman, Anthony A.; Peters, Jan-Michael

    2010-01-01

    Chromosome segregation and cell division are essential, highly ordered processes that depend on numerous protein complexes. Results from recent RNA interference (RNAi) screens indicate that the identity and composition of these protein complexes is incompletely understood. Using gene tagging on bacterial artificial chromosomes, protein localization and tandem affinity purification-mass spectrometry, the MitoCheck consortium has analyzed about 100 human protein complexes, many of which had not or only incompletely been characterized. This work has led to the discovery of previously unknown, evolutionarily conserved subunits of the anaphase-promoting complex (APC/C) and the γ-tubulin ring complex (γ-TuRC), large complexes which are essential for spindle assembly and chromosome segregation. The approaches we describe here are generally applicable to high throughput follow-up analyses of phenotypic screens in mammalian cells. PMID:20360068

  1. Systematic analysis of human protein complexes identifies chromosome segregation proteins.

    PubMed

    Hutchins, James R A; Toyoda, Yusuke; Hegemann, Björn; Poser, Ina; Hériché, Jean-Karim; Sykora, Martina M; Augsburg, Martina; Hudecz, Otto; Buschhorn, Bettina A; Bulkescher, Jutta; Conrad, Christian; Comartin, David; Schleiffer, Alexander; Sarov, Mihail; Pozniakovsky, Andrei; Slabicki, Mikolaj Michal; Schloissnig, Siegfried; Steinmacher, Ines; Leuschner, Marit; Ssykor, Andrea; Lawo, Steffen; Pelletier, Laurence; Stark, Holger; Nasmyth, Kim; Ellenberg, Jan; Durbin, Richard; Buchholz, Frank; Mechtler, Karl; Hyman, Anthony A; Peters, Jan-Michael

    2010-04-30

    Chromosome segregation and cell division are essential, highly ordered processes that depend on numerous protein complexes. Results from recent RNA interference screens indicate that the identity and composition of these protein complexes is incompletely understood. Using gene tagging on bacterial artificial chromosomes, protein localization, and tandem-affinity purification-mass spectrometry, the MitoCheck consortium has analyzed about 100 human protein complexes, many of which had not or had only incompletely been characterized. This work has led to the discovery of previously unknown, evolutionarily conserved subunits of the anaphase-promoting complex and the gamma-tubulin ring complex--large complexes that are essential for spindle assembly and chromosome segregation. The approaches we describe here are generally applicable to high-throughput follow-up analyses of phenotypic screens in mammalian cells. PMID:20360068

  2. Eukaryotic LYR Proteins Interact with Mitochondrial Protein Complexes.

    PubMed

    Angerer, Heike

    2015-01-01

    In eukaryotic cells, mitochondria host ancient essential bioenergetic and biosynthetic pathways. LYR (leucine/tyrosine/arginine) motif proteins (LYRMs) of the Complex1_LYR-like superfamily interact with protein complexes of bacterial origin. Many LYR proteins function as extra subunits (LYRM3 and LYRM6) or novel assembly factors (LYRM7, LYRM8, ACN9 and FMC1) of the oxidative phosphorylation (OXPHOS) core complexes. Structural insights into complex I accessory subunits LYRM6 and LYRM3 have been provided by analyses of EM and X-ray structures of complex I from bovine and the yeast Yarrowia lipolytica, respectively. Combined structural and biochemical studies revealed that LYRM6 resides at the matrix arm close to the ubiquinone reduction site. For LYRM3, a position at the distal proton-pumping membrane arm facing the matrix space is suggested. Both LYRMs are supposed to anchor an acyl-carrier protein (ACPM) independently to complex I. The function of this duplicated protein interaction of ACPM with respiratory complex I is still unknown. Analysis of protein-protein interaction screens, genetic analyses and predicted multi-domain LYRMs offer further clues on an interaction network and adaptor-like function of LYR proteins in mitochondria. PMID:25686363

  3. 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

  4. Complex larval connectivity patterns among marine invertebrate populations

    PubMed Central

    Becker, Bonnie J.; Levin, Lisa A.; Fodrie, F. Joel; McMillan, Pat A.

    2007-01-01

    Based on the belief that marine larvae, which can spend days to months in the planktonic stage, could be transported considerable distances by ocean currents, it has long been assumed that populations of coastal species with a planktonic larval stage are demographically open and highly “connected.” Such assumptions about the connectivity of coastal populations govern approaches to managing marine resources and shape our fundamental understanding of population dynamics and evolution, yet are rarely tested directly due to the small size and high mortality of marine larvae in a physically complex environment. Here, we document a successful application of elemental fingerprinting as a tracking tool to determine sources of settled invertebrates and show that coastal mussel larvae, previously thought to be highly dispersed, can be retained within 20–30 km of their natal origin. We compare two closely related and co-occurring species, Mytilus californianus and Mytilus galloprovincialis, and determine that, despite expected similarities, they exhibit substantially different connectivity patterns. Our use of an in situ larval culturing technique overcomes the previous challenge of applying microchemical tracking methods to species with completely planktonic development. The exchange of larvae and resulting connectivities among marine populations have fundamental consequences for the evolution and ecology of species and for the management of coastal resources. PMID:17360636

  5. 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.

  6. Patterns of Neural Activity in Networks with Complex Connectivity

    NASA Astrophysics Data System (ADS)

    Solla, Sara A.

    2008-03-01

    An understanding of emergent dynamics on complex networks requires investigating the interplay between the intrinsic dynamics of the node elements and the connectivity of the network in which they are embedded. In order to address some of these questions in a specific scenario of relevance to the dynamical states of neural ensembles, we have studied the collective behavior of excitable model neurons in a network with small-world topology. The small-world network has local lattice order, but includes a number of randomly placed connections that may provide connectivity shortcuts. This topology bears a schematic resemblance to the connectivity of the cerebral cortex, in which neurons are most strongly coupled to nearby cells within fifty to a hundred micrometers, but also make projections to cells millimeters away. We find that the dynamics of this small-world network of excitable neurons depend mostly on both the density of shortcuts and the delay associated with neuronal projections. In the regime of low shortcut density, the system exhibits persistent activity in the form of propagating waves, which annihilate upon collision and are spawned anew via the re-injection of activity through shortcut connections. As the density of shortcuts reaches a critical value, the system undergoes a transition to failure. The critical shortcut density results from matching the time associated with a recurrent path through the network to an intrinsic recovery time of the individual neurons. Furthermore, if the delay associated with neuronal interactions is sufficiently long, activity reemerges above the critical density of shortcuts. The activity in this regime exhibits long, chaotic transients composed of noisy, large-amplitude population bursts.

  7. Sampling small-scale and large-scale conformational changes in proteins and molecular complexes

    NASA Astrophysics Data System (ADS)

    Yun, Mi-Ran; Mousseau, N.; Derreumaux, P.

    2007-03-01

    Sampling of small-scale and large-scale motions is important in various computational tasks, such as protein-protein docking and ligand binding. Here, we report further development and applications of the activation-relaxation technique for internal coordinate space trajectories (ARTIST). This method generates conformational moves of any complexity and size by identifying and crossing well-defined saddle points connecting energy minima. Simulations on two all-atom proteins and three protein complexes containing between 70 and 300 amino acids indicate that ARTIST opens the door to the full treatment of all degrees of freedom in dense systems such as protein-protein complexes.

  8. Arabidopsis MSI1 connects LHP1 to PRC2 complexes.

    PubMed

    Derkacheva, Maria; Steinbach, Yvonne; Wildhaber, Thomas; Mozgová, Iva; Mahrez, Walid; Nanni, Paolo; Bischof, Sylvain; Gruissem, Wilhelm; Hennig, Lars

    2013-07-17

    Polycomb group (PcG) proteins form essential epigenetic memory systems for controlling gene expression during development in plants and animals. However, the mechanism of plant PcG protein functions remains poorly understood. Here, we probed the composition and function of plant Polycomb repressive complex 2 (PRC2). This work established the fact that all known plant PRC2 complexes contain MSI1, a homologue of Drosophila p55. While p55 is not essential for the in vitro enzymatic activity of PRC2, plant MSI1 was required for the functions of the EMBRYONIC FLOWER and the VERNALIZATION PRC2 complexes including trimethylation of histone H3 Lys27 (H3K27) at the target chromatin, as well as gene repression and establishment of competence to flower. We found that MSI1 serves to link PRC2 to LIKE HETEROCHROMATIN PROTEIN 1 (LHP1), a protein that binds H3K27me3 in vitro and in vivo and is required for a functional plant PcG system. The LHP1-MSI1 interaction forms a positive feedback loop to recruit PRC2 to chromatin that carries H3K27me3. Consequently, this can provide a mechanism for the faithful inheritance of local epigenetic information through replication. PMID:23778966

  9. 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

  10. 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

  11. Solid-phase preparation of protein complexes.

    PubMed

    Pengo, Paolo; Veggiani, Gianluca; Rattanamanee, Kwanchai; Gallotta, Andrea; Beneduce, Luca; Fassina, Giorgio

    2010-01-01

    Protein-protein conjugation is usually achieved by solution phase methods requiring concentrated protein solution and post-synthetic purification steps. In this report we describe a novel continuous-flow solid-phase approach enabling the assembly of protein complexes minimizing the amount of material needed and allowing the repeated use of the same solid phase. The method exploits an immunoaffinity matrix as solid support; the matrix reversibly binds the first of the complex components while the other components are sequentially introduced, thus allowing the complex to grow while immobilized. The tethering technique employed relies on the use of the very mild synthetic conditions and fast association rates allowed by the avidin-biotin system. At the end of the assembly, the immobilized complexes can be removed from the solid support and recovered by lowering the pH of the medium. Under the conditions used for the sequential complexation and recovery, the solid phase was not damaged or irreversibly modified and could be reused without loss of binding capacity. The method was specifically designed to prepare protein complexes to be used in immunometric methods of analysis, where the immunoreactivity of each component needs to be preserved. The approach was successfully exploited for the preparation of two different immunoaffinity reagents with immunoreactivity mimicking native squamous cell carcinoma antigen-immunoglobulin M (SCCA-IgM) and alphafetoprotein-immunoglobulin M (AFP-IgM) immune complexes, which were characterized by dedicated sandwich enzyme-linked immunosorbent assay (ELISA) and immunoblot. Besides the specific application described in the paper, the method is sufficiently general to be used for the preparation of a broad range of protein assemblies. PMID:21038355

  12. Solvation dynamics in a protein surfactant complex

    NASA Astrophysics Data System (ADS)

    Dutta, Partha; Sen, Pratik; Halder, Arnab; Mukherjee, Saptarshi; Sen, Sobhan; Bhattacharyya, Kankan

    2003-08-01

    Solvation dynamics in the denatured state of a protein, lysozyme (denatured by sodium dodecyl sulfate, SDS) is markedly slower than that in the native state. For coumarin 153 bound to lysozyme, the average solvation time, < τs> is 330 ps. In the lysozyme-SDS complex, the solvation dynamics is markedly slower with < τs>=7250 ps. On addition of dithiothreitol (DTT) to the lysozyme-SDS complex, when the di-sulfide bonds are destroyed, < τs> is found to be 1140 ps. The slow dynamics in the denatured protein is attributed to the polymer chain dynamics and the exchange of bound and free water molecules.

  13. 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

  14. Metal complexes as "protein surface mimetics".

    PubMed

    Hewitt, Sarah H; Wilson, Andrew J

    2016-07-28

    A key challenge in chemical biology is to identify small molecule regulators for every single protein. However, protein surfaces are notoriously difficult to recognise with synthetic molecules, often having large flat surfaces that are poorly matched to traditional small molecules. In the surface mimetic approach, a supramolecular scaffold is used to project recognition groups in such a manner as to make multivalent non-covalent contacts over a large area of protein surface. Metal based supramolecular scaffolds offer unique advantages over conventional organic molecules for protein binding, including greater stereochemical and geometrical diversity conferred through the metal centre and the potential for direct assessment of binding properties and even visualisation in cells without recourse to further functionalisation. This feature article will highlight the current state of the art in protein surface recognition using metal complexes as surface mimetics. PMID:27353704

  15. 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

  16. Assembly reflects evolution of protein complexes.

    PubMed

    Levy, Emmanuel D; Boeri Erba, Elisabetta; Robinson, Carol V; Teichmann, Sarah A

    2008-06-26

    A homomer is formed by self-interacting copies of a protein unit. This is functionally important, as in allostery, and structurally crucial because mis-assembly of homomers is implicated in disease. Homomers are widespread, with 50-70% of proteins with a known quaternary state assembling into such structures. Despite their prevalence, their role in the evolution of cellular machinery and the potential for their use in the design of new molecular machines, little is known about the mechanisms that drive formation of homomers at the level of evolution and assembly in the cell. Here we present an analysis of over 5,000 unique atomic structures and show that the quaternary structure of homomers is conserved in over 70% of protein pairs sharing as little as 30% sequence identity. Where quaternary structure is not conserved among the members of a protein family, a detailed investigation revealed well-defined evolutionary pathways by which proteins transit between different quaternary structure types. Furthermore, we show by perturbing subunit interfaces within complexes and by mass spectrometry analysis, that the (dis)assembly pathway mimics the evolutionary pathway. These data represent a molecular analogy to Haeckel's evolutionary paradigm of embryonic development, where an intermediate in the assembly of a complex represents a form that appeared in its own evolutionary history. Our model of self-assembly allows reliable prediction of evolution and assembly of a complex solely from its crystal structure. PMID:18563089

  17. 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.

  18. Radiolysis of DNA-protein complexes

    NASA Astrophysics Data System (ADS)

    Běgusová, Marie; Gillard, Nathalie; Sy, Denise; Castaing, Bertrand; Charlier, Michel; Spotheim-Maurizot, Melanie

    2005-02-01

    We discuss here modifications of DNA and protein radiolysis due to the interaction of these two partners in specific complexes. Experimental patterns of frank strand breaks (FSB) and alkali revealed breaks (ARB) obtained for DNA lac operator bound to the lac repressor and for a DNA containing an abasic site analog bound to the formamidopyrimidine-DNA glycosylase are reported. Experimental data are compared to predicted damage distribution obtained using the theoretical model RADACK.

  19. 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

  20. Gene Duplicability-Connectivity-Complexity across Organisms and a Neutral Evolutionary Explanation

    PubMed Central

    Zhu, Yun; Du, Peng; Nakhleh, Luay

    2012-01-01

    Gene duplication has long been acknowledged by biologists as a major evolutionary force shaping genomic architectures and characteristics across the Tree of Life. Major research has been conducting on elucidating the fate of duplicated genes in a variety of organisms, as well as factors that affect a gene’s duplicability–that is, the tendency of certain genes to retain more duplicates than others. In particular, two studies have looked at the correlation between gene duplicability and its degree in a protein-protein interaction network in yeast, mouse, and human, and another has looked at the correlation between gene duplicability and its complexity (length, number of domains, etc.) in yeast. In this paper, we extend these studies to six species, and two trends emerge. There is an increase in the duplicability-connectivity correlation that agrees with the increase in the genome size as well as the phylogenetic relationship of the species. Further, the duplicability-complexity correlation seems to be constant across the species. We argue that the observed correlations can be explained by neutral evolutionary forces acting on the genomic regions containing the genes. For the duplicability-connectivity correlation, we show through simulations that an increasing trend can be obtained by adjusting parameters to approximate genomic characteristics of the respective species. Our results call for more research into factors, adaptive and non-adaptive alike, that determine a gene’s duplicability. PMID:22984517

  1. 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.

  2. Dynamics connect substrate recognition to catalysis in protein kinase A

    PubMed Central

    Masterson, Larry R.; Cheng, Cecilia; Yu, Tao; Tonelli, Marco; Kornev, Alexandr; Taylor, Susan S.; Veglia, Gianluigi

    2012-01-01

    Atomic resolution studies of protein kinases have traditionally been carried out in the inhibitory state, limiting our current knowledge on the mechanisms of substrate recognition and catalysis. Using NMR, x-ray crystallography, and thermodynamic measurements we analyzed the substrate recognition process of cAMP-dependent protein kinase (PKA), finding that entropy and protein dynamics play a prominent role. The nucleotide acts as a dynamic and allosteric activator by coupling the two lobes of apo PKA, enhancing the enzyme dynamics synchronously, and priming it for catalysis. The formation of the ternary complex is entropically driven and NMR spin relaxation data reveal that both substrate and PKA are dynamic in the closed state. Our results show that the enzyme toggles between open and closed states, which indicate that a population shift/conformational selection rather than an induced-fit mechanism governs substrate recognition. PMID:20890288

  3. 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

  4. A Least Square Method Based Model for Identifying Protein Complexes in Protein-Protein Interaction Network

    PubMed Central

    Dai, Qiguo; Guo, Maozu; Guo, Yingjie; Liu, Xiaoyan; Liu, Yang; Teng, Zhixia

    2014-01-01

    Protein complex formed by a group of physical interacting proteins plays a crucial role in cell activities. Great effort has been made to computationally identify protein complexes from protein-protein interaction (PPI) network. However, the accuracy of the prediction is still far from being satisfactory, because the topological structures of protein complexes in the PPI network are too complicated. This paper proposes a novel optimization framework to detect complexes from PPI network, named PLSMC. The method is on the basis of the fact that if two proteins are in a common complex, they are likely to be interacting. PLSMC employs this relation to determine complexes by a penalized least squares method. PLSMC is applied to several public yeast PPI networks, and compared with several state-of-the-art methods. The results indicate that PLSMC outperforms other methods. In particular, complexes predicted by PLSMC can match known complexes with a higher accuracy than other methods. Furthermore, the predicted complexes have high functional homogeneity. PMID:25405206

  5. 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

  6. 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.…

  7. 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

  8. 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

  9. 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.

  10. 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.

  11. The Response of Greek Key Proteins to Changes in Connectivity Depends on the Nature of Their Secondary Structure

    PubMed Central

    Kemplen, Katherine R.; De Sancho, David; Clarke, Jane

    2015-01-01

    What governs the balance between connectivity and topology in regulating the mechanism of protein folding? We use circular permutation to vary the order of the helices in the all-α Greek key protein FADD (Fas-associated death domain) to investigate this question. Unlike all-β Greek key proteins, where changes in the order of secondary structure cause a shift in the folding nucleus, the position of the nucleus in FADD is unchanged, even when permutation reduces the complexity significantly. We suggest that this is because local helical contacts are so dominant that permutation has little effect on the entropic cost of forming the folding nucleus whereas, in all-β Greek key proteins, all interactions in the nucleus are long range. Thus, the type of secondary structure modulates the sensitivity of proteins to changes in connectivity. PMID:25861761

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

    PubMed Central

    Zaki, Nazar; Mohamed, Elfadil A.; Mora, Antonio

    2015-01-01

    The identification and characterization of protein complexes implicated in protein-protein interaction data are crucial to the understanding of the molecular events under normal and abnormal physiological conditions. This paper provides a novel characterization of subcomplexes in protein interaction databases, stressing definition and representation issues, quantification, biological validation, network metrics, motifs, modularity, and gene ontology (GO) terms. The paper introduces the concept of “nested group” as a way to represent subcomplexes and estimates that around 15% of those nested group with the higher Jaccard index may be a result of data artifacts in protein interaction databases, while a number of them can be found in biologically important modular structures or dynamic structures. We also found that network centralities, enrichment in essential proteins, GO terms related to regulation, imperfect 5-clique motifs, and higher GO homogeneity can be used to identify proteins in nested complexes. PMID:25722891

  13. Identification and analysis of multi-protein complexes in placenta.

    PubMed

    Wang, Fuqiang; Wang, Ling; Xu, Zhiyang; Liang, Gaolin

    2013-01-01

    Placental malfunction induces pregnancy disorders which contribute to life-threatening complications for both the mother and the fetus. Identification and characterization of placental multi-protein complexes is an important step to integratedly understand the protein-protein interaction networks in placenta which determine placental function. In this study, blue native/sodium dodecyl sulfate polyacrylamide gel electrophoresis (BN/SDS-PAGE) and Liquid chromatography-tandem mass spectrometry (LC-MS/MS) were used to screen the multi-protein complexes in placenta. 733 unique proteins and 34 known and novel heterooligomeric multi-protein complexes including mitochondrial respiratory chain complexes, integrin complexes, proteasome complexes, histone complex, and heat shock protein complexes were identified. A novel protein complex, which involves clathrin and small conductance calcium-activated potassium (SK) channel protein 2, was identified and validated by antibody based gel shift assay, co-immunoprecipitation and immunofluorescence staining. These results suggest that BN/SDS-PAGE, when integrated with LC-MS/MS, is a very powerful and versatile tool for the investigation of placental protein complexes. This work paves the way for deeper functional characterization of the placental protein complexes associated with pregnancy disorders. PMID:23638173

  14. 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

  15. 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

  16. 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. PMID:24642838

  17. Detecting Overlapping Protein Complexes by Rough-Fuzzy Clustering in Protein-Protein Interaction Networks

    PubMed Central

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

    2014-01-01

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

  18. Assessing small-worldness of dynamic functional brain connectivity during complex tasks.

    PubMed

    Shen Ren; Taya, Fumihiko; Yu Sun; deSouza, Joshua; Thakor, Nitish V; Bezerianos, Anastasios

    2015-08-01

    The development of network theory has introduced new approaches to understand the brain as a complex system. Currently the time-variant functional connectivity of brain networks under complex tasks is still being investigated. To explore connectivity during complex cognitive and motor tasks, this study focused on the relevance of small-worldness to human workloads using EEG signals from a dynamic analytic approach. Experiments were designed to investigate the small-worldness under two types of flight simulation tasks at two levels of difficulty - easy and hard. The results demonstrated a consistent small-world architecture of brain connectivity with time-based variance during complex tasks. We noticed an increased small-world effect especially at the alpha band when performing hard tasks compared to easy tasks, which relate to high and low workload respectively. Our results show the potential of dynamic brain network analysis in exploring time-variant and task-dependent brain connectivity during complex tasks. PMID:26736899

  19. The pain interactome: Connecting pain-specific protein interactions

    PubMed Central

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

    2014-01-01

    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. PMID:24978826

  20. 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

  1. 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

  2. Magnetic Resonance Access to Transiently Formed Protein Complexes**

    PubMed Central

    Sára, Tomáš; Schwarz, Thomas C; Kurzbach, Dennis; Wunderlich, Christoph H; Kreutz, Christoph; Konrat, Robert

    2014-01-01

    Protein–protein interactions are of utmost importance to an understanding of biological phenomena since non-covalent and therefore reversible couplings between basic proteins leads to the formation of complex regulatory and adaptive molecular systems. Such systems are capable of maintaining their integrity and respond to external stimuli, processes intimately related to living organisms. These interactions, however, span a wide range of dissociation constants, from sub-nanomolar affinities in tight complexes to high-micromolar or even millimolar affinities in weak, transiently formed protein complexes. Herein, we demonstrate how novel NMR and EPR techniques can be used for the characterization of weak protein–protein (ligand) complexes. Applications to intrinsically disordered proteins and transiently formed protein complexes illustrate the potential of these novel techniques to study hitherto unobserved (and unobservable) higher-order structures of proteins. PMID:25050230

  3. 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

  4. An overview of the structures of protein-DNA complexes

    PubMed Central

    Luscombe, Nicholas M; Austin, Susan E; Berman , Helen M; Thornton, Janet M

    2000-01-01

    On the basis of a structural analysis of 240 protein-DNA complexes contained in the Protein Data Bank (PDB), we have classified the DNA-binding proteins involved into eight different structural/functional groups, which are further classified into 54 structural families. Here we present this classification and review the functions, structures and binding interactions of these protein-DNA complexes. PMID:11104519

  5. 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…

  6. Connecting core percolation and controllability of complex networks.

    PubMed

    Jia, Tao; Pósfai, Márton

    2014-01-01

    Core percolation is a fundamental structural transition in complex networks related to a wide range of important problems. Recent advances have provided us an analytical framework of core percolation in uncorrelated random networks with arbitrary degree distributions. Here we apply the tools in analysis of network controllability. We confirm analytically that the emergence of the bifurcation in control coincides with the formation of the core and the structure of the core determines the control mode of the network. We also derive the analytical expression related to the controllability robustness by extending the deduction in core percolation. These findings help us better understand the interesting interplay between the structural and dynamical properties of complex networks. PMID:24946797

  7. The multifaceted roles of intrinsic disorder in protein complexes.

    PubMed

    Uversky, Vladimir N

    2015-09-14

    Intrinsically disordered proteins (IDPs) and intrinsically disordered protein regions (IDPRs) are important constituents of many protein complexes, playing various structural, functional, and regulatory roles. In such disorder-based protein complexes, functional disorder is used both internally (for assembly, movement, and functional regulation of the different parts of a given complex) and externally (for interactions of a complex with its external regulators). In complex assembly, IDPs/IDPRs serve as the molecular glue that cements complexes or as highly flexible scaffolds. Disorder defines the order of complex assembly and the ability of a protein to be involved in polyvalent interactions. It is at the heart of various binding mechanisms and interaction modes ascribed to IDPs. Disorder in protein complexes is related to multifarious applications of induced folding and induced functional unfolding, or defines the entropic chain activities, such as stochastic machines and binding rheostats. This review opens a FEBS Letters Special Issue on Dynamics, Flexibility, and Intrinsic Disorder in protein assemblies and represents a brief overview of intricate roles played by IDPs and IDPRs in various aspects of protein complexes. PMID:26073257

  8. Chlorophyll-Protein Complexes of the Cyanophyte, Nostoc sp. 1

    PubMed Central

    Rusckowski, Mary; Zilinskas, Barbara A.

    1980-01-01

    Four chlorophyll-protein complexes have been resolved from the cyanophyte, Nostoc sp., by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis at 4 C. Complexes solubilized by SDS from Spinacia oleracea were run for comparison. As has been well documented, the P700-chlorophyll a-protein complex from the higher plant and blue-green algal samples are similar, and the light-harvesting pigment protein complex is present only in the former. Most noteworthy are two closely migrating chlorophyll proteins in Nostoc sp. which have approximately the same mobility as a single chlorophyll-protein band resolvable from spinach. The absorption maximum of the complex from spinach is at 667 nanometers, and those of the two complexes from Nostoc sp. are at 667 and 669 nanometers; the fluorescence emission maximum at −196 C is at 685 nanometers, and the 735 nanometer fluorescence peak, characteristic of the P700-chlorophyll a-protein complex, is absent. The apoproteins of these new complexes from Nostoc sp. and spinach are in the kilodalton range. It appears that at least one of these two chlorophyll-protein complexes from Nostoc sp. compares with those recently described by others from higher plants and green algae as likely photosystem II complexes, perhaps containing P680, although no photochemical data are yet available. Images PMID:16661198

  9. Hysteresis as a Marker for Complex, Overlapping Landscapes in Proteins

    PubMed Central

    Andrews, Benjamin T.; Capraro, Dominique T.; Sulkowska, Joanna I.; Onuchic, José N.; Jennings, Patricia A.

    2013-01-01

    Topologically complex proteins fold by multiple routes as a result of hard-to-fold regions of the proteins. Oftentimes these regions are introduced into the protein scaffold for function and increase frustration in the otherwise smooth-funneled landscape. Interestingly, while functional regions add complexity to folding landscapes, they may also contribute to a unique behavior referred to as hysteresis. While hysteresis is predicted to be rare, it is observed in various proteins, including proteins containing a unique peptide cyclization to form a fluorescent chromophore as well as proteins containing a knotted topology in their native fold. Here, hysteresis is demonstrated to be a consequence of the decoupling of unfolding events from the isomerization or hula-twist of a chromophore in one protein and the untying of the knot in a second protein system. The question now is- can hysteresis be a marker for the interplay of landscapes where complex folding and functional regions overlap? PMID:23525263

  10. Advances in protein complex analysis using mass spectrometry.

    PubMed

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

    2005-02-15

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

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

    PubMed

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

    2010-10-01

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

  12. Global landscape of protein complexes in the yeast Saccharomyces cerevisiae.

    PubMed

    Krogan, Nevan J; Cagney, Gerard; Yu, Haiyuan; Zhong, Gouqing; Guo, Xinghua; Ignatchenko, Alexandr; Li, Joyce; Pu, Shuye; Datta, Nira; Tikuisis, Aaron P; Punna, Thanuja; Peregrín-Alvarez, José M; Shales, Michael; Zhang, Xin; Davey, Michael; Robinson, Mark D; Paccanaro, Alberto; Bray, James E; Sheung, Anthony; Beattie, Bryan; Richards, Dawn P; Canadien, Veronica; Lalev, Atanas; Mena, Frank; Wong, Peter; Starostine, Andrei; Canete, Myra M; Vlasblom, James; Wu, Samuel; Orsi, Chris; Collins, Sean R; Chandran, Shamanta; Haw, Robin; Rilstone, Jennifer J; Gandi, Kiran; Thompson, Natalie J; Musso, Gabe; St Onge, Peter; Ghanny, Shaun; Lam, Mandy H Y; Butland, Gareth; Altaf-Ul, Amin M; Kanaya, Shigehiko; Shilatifard, Ali; O'Shea, Erin; Weissman, Jonathan S; Ingles, C James; Hughes, Timothy R; Parkinson, John; Gerstein, Mark; Wodak, Shoshana J; Emili, Andrew; Greenblatt, Jack F

    2006-03-30

    Identification of protein-protein interactions often provides insight into protein function, and many cellular processes are performed by stable protein complexes. We used tandem affinity purification to process 4,562 different tagged proteins of the yeast Saccharomyces cerevisiae. Each preparation was analysed by both matrix-assisted laser desorption/ionization-time of flight mass spectrometry and liquid chromatography tandem mass spectrometry to increase coverage and accuracy. Machine learning was used to integrate the mass spectrometry scores and assign probabilities to the protein-protein interactions. Among 4,087 different proteins identified with high confidence by mass spectrometry from 2,357 successful purifications, our core data set (median precision of 0.69) comprises 7,123 protein-protein interactions involving 2,708 proteins. A Markov clustering algorithm organized these interactions into 547 protein complexes averaging 4.9 subunits per complex, about half of them absent from the MIPS database, as well as 429 additional interactions between pairs of complexes. The data (all of which are available online) will help future studies on individual proteins as well as functional genomics and systems biology. PMID:16554755

  13. Antifreeze Proteins in Winter Rye Leaves Form Oligomeric Complexes1

    PubMed Central

    Yu, Xiao-Ming; Griffith, Marilyn

    1999-01-01

    Antifreeze proteins (AFPs) similar to three pathogenesis-related proteins, a glucanase-like protein (GLP), a chitinase-like protein (CLP), and a thaumatin-like protein (TLP), accumulate during cold acclimation in winter rye (Secale cereale) leaves, where they are thought to modify the growth of intercellular ice during freezing. The objective of this study was to characterize the rye AFPs in their native forms, and our results show that these proteins form oligomeric complexes in vivo. Nine proteins were separated by native-polyacrylamide gel electrophoresis from apoplastic extracts of cold-acclimated winter rye leaves. Seven of these proteins exhibited multiple polypeptides when denatured and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. After isolation of the individual proteins, six were shown by immunoblotting to contain various combinations of GLP, CLP, and TLP in addition to other unidentified proteins. Antisera produced against individual cold-induced winter rye GLP, CLP, and TLP all dramatically inhibited glucanase activity in apoplastic extracts from cold-acclimated winter rye leaves, and each antiserum precipitated all three proteins. These results indicate that each of the polypeptides may be exposed on the surface of the protein complexes. By forming oligomeric complexes, AFPs may form larger surfaces to interact with ice, or they may simply increase the mass of the protein bound to ice. In either case, the complexes of AFPs may inhibit ice growth and recrystallization more effectively than the individual polypeptides. PMID:10198095

  14. 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

  15. 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

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

    PubMed Central

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

    2010-01-01

    Protein-protein interactions are important for nearly all biological processes, and it is known that aberrant protein-protein interactions can lead to human disease and cancer. Recent evidence has suggested that protein interaction interfaces describe a new class of attractive targets for drug development. Full characterization of protein interaction networks of protein complexes and their dynamics in response to various cellular cues will provide essential information for us to understand how protein complexes work together in cells to maintain cell viability and normal homeostasis. Affinity purification coupled with quantitative mass spectrometry has become the primary method for studying in vivo protein interactions of protein complexes and whole organism proteomes. Recent developments in sample preparation and affinity purification strategies allow the capture, identification, and quantification of protein interactions of protein complexes that are stable, dynamic, transient, and/or weak. Current efforts have mainly focused on generating reliable, reproducible, and high confidence protein interaction data sets for functional characterization. The availability of increasing amounts of information on protein interactions in eukaryotic systems and new bioinformatics tools allow functional analysis of quantitative protein interaction data to unravel the biological significance of the identified protein interactions. Existing studies in this area have laid a solid foundation toward generating a complete map of in vivo protein interaction networks of protein complexes in cells or tissues. PMID:20445003

  17. 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.

  18. 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

  19. 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

  20. Multiscale Model for the Assembly Kinetics of Protein Complexes.

    PubMed

    Xie, Zhong-Ru; Chen, Jiawen; Wu, Yinghao

    2016-02-01

    The assembly of proteins into high-order complexes is a general mechanism for these biomolecules to implement their versatile functions in cells. Natural evolution has developed various assembling pathways for specific protein complexes to maintain their stability and proper activities. Previous studies have provided numerous examples of the misassembly of protein complexes leading to severe biological consequences. Although the research focusing on protein complexes has started to move beyond the static representation of quaternary structures to the dynamic aspect of their assembly, the current understanding of the assembly mechanism of protein complexes is still largely limited. To tackle this problem, we developed a new multiscale modeling framework. This framework combines a lower-resolution rigid-body-based simulation with a higher-resolution Cα-based simulation method so that protein complexes can be assembled with both structural details and computational efficiency. We applied this model to a homotrimer and a heterotetramer as simple test systems. Consistent with experimental observations, our simulations indicated very different kinetics between protein oligomerization and dimerization. The formation of protein oligomers is a multistep process that is much slower than dimerization but thermodynamically more stable. Moreover, we showed that even the same protein quaternary structure can have very diverse assembly pathways under different binding constants between subunits, which is important for regulating the functions of protein complexes. Finally, we revealed that the binding between subunits in a complex can be synergistically strengthened during assembly without considering allosteric regulation or conformational changes. Therefore, our model provides a useful tool to understand the general principles of protein complex assembly. PMID:26738810

  1. SnapShot: SMC Protein Complexes Part II.

    PubMed

    Haering, Christian H; Gruber, Stephan

    2016-02-11

    This second of two SnapShots on SMC proteins depicts their roles at different stages of the eukaryotic cell cycle. The composition and architecture of SMC protein complexes and their regulators appear in SMC Protein Complexes Part I (available at http://www.cell.com/cell/pdf/S0092-8674%2815%2901690-6.pdf). To view this SnapShot, open or download the PDF. PMID:26871638

  2. Identifying protein complexes in protein-protein interaction networks by using clique seeds and graph entropy.

    PubMed

    Chen, Bolin; Shi, Jinhong; Zhang, Shenggui; Wu, Fang-Xiang

    2013-01-01

    The identification of protein complexes plays a key role in understanding major cellular processes and biological functions. Various computational algorithms have been proposed to identify protein complexes from protein-protein interaction (PPI) networks. In this paper, we first introduce a new seed-selection strategy for seed-growth style algorithms. Cliques rather than individual vertices are employed as initial seeds. After that, a result-modification approach is proposed based on this seed-selection strategy. Predictions generated by higher order clique seeds are employed to modify results that are generated by lower order ones. The performance of this seed-selection strategy and the result-modification approach are tested by using the entropy-based algorithm, which is currently the best seed-growth style algorithm to detect protein complexes from PPI networks. In addition, we investigate four pairs of strategies for this algorithm in order to improve its accuracy. The numerical experiments are conducted on a Saccharomyces cerevisiae PPI network. The group of best predictions consists of 1711 clusters, with the average f-score at 0.68 after removing all similar and redundant clusters. We conclude that higher order clique seeds can generate predictions with higher accuracy and that our improved entropy-based algorithm outputs more reasonable predictions than the original one. PMID:23112006

  3. 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

  4. 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

  5. The Responsive Amygdala: Treatment-induced Alterations in Functional Connectivity in Pediatric Complex Regional Pain Syndrome

    PubMed Central

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

    2014-01-01

    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-gender matched controls 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 to controls, with differences predominantly in the left amygdala in the pre-treated 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 controls 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 following an aggressive treatment program in children with chronic pain and intrinsic amygdala functional connectivity activity serving as a potential indicator of treatment response. PMID:24861582

  6. 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. PMID:24861582

  7. 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.

  8. U1-RNP and TLR receptors in the pathogenesis of mixed connective tissue diseasePart I. The U1-RNP complex and its biological significance in the pathogenesis of mixed connective tissue disease

    PubMed Central

    2015-01-01

    Mixed connective tissue disease (MCTD) is a rare autoimmune syndrome, signified by complex interactions between disease-related phenomena, including inflammation, proliferative vascular arteriopathy, thrombotic events and humoral autoimmune processes. It is still controversial whether MCTD is a distinct clinical entity among systemic connective tissue diseases, although several authors consider that it is distinct and underline characteristic, distinct clinical, serological and immunogenetic features. The putative target of autoimmunity in MCTD is U1-RNP, which is a complex of U1-RNA and small nuclear RNP. Both the U1-RNA component and the specific proteins, particularly U1-70K, engage immune cells and their receptors in a complex network of interactions that ultimately lead to autoimmunity, inflammation, and tissue injury. U1-RNA is capable of inducing manifestations consistent with TLR activation. Stimulation of innate immunity by native RNA molecules with a double-stranded secondary structure may help explain the high prevalence of autoimmunity to RNA binding proteins.

  9. 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.

  10. Proteomic comparison of etioplast and chloroplast protein complexes.

    PubMed

    Plöscher, Matthias; Reisinger, Veronika; Eichacker, Lutz A

    2011-08-12

    Angiosperms grown in darkness develop etioplasts during skotomorphogenesis. It is well known that etioplasts accumulate large quantities of protochlorophyllideoxidoreductase, are devoid of chlorophyll and are the site to assemble the photosynthetic machinery during photomorphogenesis. Proteomic investigation of the membrane protein complexes by Native PAGE, in combination with CyDye labelling and mass spectrometric analysis revealed that etioplasts and chloroplasts share a number of membrane protein complexes characteristic for electron transport, chlorophyll and protein synthesis as well as fatty acid biosynthesis. The complex regulatory function in both developmental states is discussed. PMID:21440687

  11. 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

  12. 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.

  13. Transient and persistent pain induced connectivity alterations in pediatric complex regional pain syndrome.

    PubMed

    Linnman, Clas; Becerra, Lino; Lebel, Alyssa; Berde, Charles; Grant, P Ellen; Borsook, David

    2013-01-01

    Evaluation of pain-induced changes in functional connectivity was performed in pediatric complex regional pain syndrome (CRPS) patients. High field functional magnetic resonance imaging was done in the symptomatic painful state and at follow up in the asymptomatic pain free/recovered state. Two types of connectivity alterations were defined: (1) Transient increases in functional connectivity that identified regions with increased cold-induced functional connectivity in the affected limb vs. unaffected limb in the CRPS state, but with normalized connectivity patterns in the recovered state; and (2) Persistent increases in functional connectivity that identified regions with increased cold-induced functional connectivity in the affected limb as compared to the unaffected limb that persisted also in the recovered state (recovered affected limb versus recovered unaffected limb). The data support the notion that even after symptomatic recovery, alterations in brain systems persist, particularly in amygdala and basal ganglia systems. Connectivity analysis may provide a measure of temporal normalization of different circuits/regions when evaluating therapeutic interventions for this condition. The results add emphasis to the importance of early recognition and management in improving outcome of pediatric CRPS. PMID:23526938

  14. Transient and Persistent Pain Induced Connectivity Alterations in Pediatric Complex Regional Pain Syndrome

    PubMed Central

    Linnman, Clas; Becerra, Lino; Lebel, Alyssa; Berde, Charles; Grant, P. Ellen; Borsook, David

    2013-01-01

    Evaluation of pain-induced changes in functional connectivity was performed in pediatric complex regional pain syndrome (CRPS) patients. High field functional magnetic resonance imaging was done in the symptomatic painful state and at follow up in the asymptomatic pain free/recovered state. Two types of connectivity alterations were defined: (1) Transient increases in functional connectivity that identified regions with increased cold-induced functional connectivity in the affected limb vs. unaffected limb in the CRPS state, but with normalized connectivity patterns in the recovered state; and (2) Persistent increases in functional connectivity that identified regions with increased cold-induced functional connectivity in the affected limb as compared to the unaffected limb that persisted also in the recovered state (recovered affected limb versus recovered unaffected limb). The data support the notion that even after symptomatic recovery, alterations in brain systems persist, particularly in amygdala and basal ganglia systems. Connectivity analysis may provide a measure of temporal normalization of different circuits/regions when evaluating therapeutic interventions for this condition. The results add emphasis to the importance of early recognition and management in improving outcome of pediatric CRPS. PMID:23526938

  15. 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. PMID:25536493

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

    PubMed Central

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

    2015-01-01

    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. PMID:25536493

  17. 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.

  18. Sizing Large Proteins and Protein Complexes by Electrospray Ionization Mass Spectrometry and Ion Mobility

    PubMed Central

    Kaddis, Catherine S.; Lomeli, Shirley H.; Yin, Sheng; Berhane, Beniam; Apostol, Marcin I.; Kickhoefer, Valerie A.; Rome, Leonard H.; Loo, Joseph A.

    2009-01-01

    Mass spectrometry (MS) and ion mobility with electrospray ionization (ESI) have the capability to measure and detect large noncovalent protein-ligand and protein-protein complexes. Using an ion mobility method termed GEMMA (Gas-Phase Electrophoretic Mobility Molecular Analysis), protein particles representing a range of sizes can be separated by their electrophoretic mobility in air. Highly charged particles produced from a protein complex solution using electrospray can be manipulated to produce singly charged ions which can be separated and quantified by their electrophoretic mobility. Results from ESI-GEMMA analysis from our laboratory and others were compared to other experimental and theoretically determined parameters, such as molecular mass and cryoelectron microscopy and x-ray crystal structure dimensions. There is a strong correlation between the electrophoretic mobility diameter determined from GEMMA analysis and the molecular mass for protein complexes up to 12 MDa, including the 93 kDa enolase dimer, the 480 kDa ferritin 24-mer complex, the 4.6 MDa cowpea chlorotic mottle virus (CCMV), and the 9 MDa MVP-vault assembly. ESI-GEMMA is used to differentiate a number of similarly sized vault complexes that are composed of different N-terminal protein tags on the MVP subunit. The average effective density of the proteins and protein complexes studied was 0.6 g/cm3. Moreover, there is evidence that proteins and protein complexes collapse or become more compact in the gas phase in the absence of water. PMID:17434746

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

    SciTech Connect

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

    2005-04-11

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

  20. 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.…

  1. Protein structures in SDS micelle-protein complexes.

    PubMed Central

    Parker, W; Song, P S

    1992-01-01

    Sodium dodecyl sulfate (SDS) is used more often than any other detergent as an excellent denaturing or "unfolding" detergent. However, formation of ordered structure (alpha-helix or beta-sheet) in certain peptides is known to be induced by interaction with SDS micelles. The SDS-induced structures formed by these peptides are amphiphilic, having both a hydrophobic and a hydrophilic face. Previous work in this area has revealed that SDS induces helical folding in a wide variety of non-helical proteins. Here, we describe the interaction of several structurally unrelated proteins with SDS micelles and the correlation of these structures to helical amphiphilic regions present in the primary sequence. It is likely that the ability of native nonordered protein structures to form induced amphiphilic ordered structures is rather common. PMID:1600087

  2. 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

  3. PEGylated Albumin-Based Polyion Complex Micelles for Protein Delivery.

    PubMed

    Jiang, Yanyan; Lu, Hongxu; Chen, Fan; Callari, Manuela; Pourgholami, Mohammad; Morris, David L; Stenzel, Martina H

    2016-03-14

    An increasing amount of therapeutic agents are based on proteins. However, proteins as drug have intrinsic problems such as their low hydrolytic stability. Delivery of proteins using nanoparticles has increasingly been the focus of interest with polyion complex micelles, prepared from charged block copolymer and the oppositely charged protein, as an example of an attractive carrier for proteins. Inspired by this approach, a more biocompatible pathway has been developed here, which replaces the charged synthetic polymer with an abundant protein, such as albumin. Although bovine serum albumin (BSA) was observed to form complexes with positively charged proteins directly, the resulting protein nanoparticle were not stable and aggregated to large precipitates over the course of a day. Therefore, maleimide functionalized poly(oligo (ethylene glycol) methyl ether methacrylate) (MI-POEGMEMA) (Mn = 26000 g/mol) was synthesized to generate a polymer-albumin conjugate, which was able to condense positively charged proteins, here lysozyme (Lyz) as a model. The PEGylated albumin polyion complex micelle with lysozyme led to nanoparticles between 15 and 25 nm in size depending on the BSA to Lyz ratio. The activity of the encapsulated protein was tested using Sprouty 1 (C-12; Spry1) proteins, which can act as an endogenous angiogenesis inhibitor. Condensation of Spry1 with the PEGylated albumin could improve the anticancer efficacy of Spry1 against the breast cancer cells lowering the IC50 value of the protein. Furthermore, the high anticancer efficacy of the POEGMEMA-BSA/Spry1 complex micelle was verified by effectively inhibiting the growth of three-dimensional MCF-7 multicellular tumor spheroids. The PEGylated albumin complex micelle has great potential as a drug delivery vehicle for a new generation of cancer pharmaceuticals. PMID:26809948

  4. 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

  5. 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. PMID:21036361

  6. 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

  7. 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…

  8. Cross-linking Measurements of In Vivo Protein Complex Topologies*

    PubMed Central

    Zheng, Chunxiang; Yang, Li; Hoopmann, Michael R.; Eng, Jimmy K.; Tang, Xiaoting; Weisbrod, Chad R.; Bruce, James E.

    2011-01-01

    Identification and measurement of in vivo protein interactions pose critical challenges in the goal to understand biological systems. The measurement of structures and topologies of proteins and protein complexes as they exist in cells is particularly challenging, yet critically important to improve understanding of biological function because proteins exert their intended function only through the structures and interactions they exhibit in vivo. In the present study, protein interactions in E. coli cells were identified in our unbiased cross-linking approach, yielding the first in vivo topological data on many interactions and the largest set of identified in vivo cross-linked peptides produced to date. These data show excellent agreement with protein and complex crystal structures where available. Furthermore, our unbiased data provide novel in vivo topological information that can impact understanding of biological function, even for cases where high resolution structures are not yet available. PMID:21697552

  9. Graph theory and stability analysis of protein complex interaction networks.

    PubMed

    Huang, Chien-Hung; Chen, Teng-Hung; Ng, Ka-Lok

    2016-04-01

    Protein complexes play an essential role in many biological processes. Complexes can interact with other complexes to form protein complex interaction network (PCIN) that involves in important cellular processes. There are relatively few studies on examining the interaction topology among protein complexes; and little is known about the stability of PCIN under perturbations. We employed graph theoretical approach to reveal hidden properties and features of four species PCINs. Two main issues are addressed, (i) the global and local network topological properties, and (ii) the stability of the networks under 12 types of perturbations. According to the topological parameter classification, we identified some critical protein complexes and validated that the topological analysis approach could provide meaningful biological interpretations of the protein complex systems. Through the Kolmogorov-Smimov test, we showed that local topological parameters are good indicators to characterise the structure of PCINs. We further demonstrated the effectiveness of the current approach by performing the scalability and data normalization tests. To measure the robustness of PCINs, we proposed to consider eight topological-based perturbations, which are specifically applicable in scenarios of targeted, sustained attacks. We found that the degree-based, betweenness-based and brokering-coefficient-based perturbations have the largest effect on network stability. PMID:26997661

  10. 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

  11. SnapShot: SMC Protein Complexes Part I.

    PubMed

    Haering, Christian H; Gruber, Stephan

    2016-01-14

    This first of two SnapShots on SMC proteins depicts the composition and architecture of SMC protein complexes and their regulators. Their roles at different stages of the cell cycle will appear in Part II. To view this SnapShot, open or download the PDF. PMID:26771499

  12. Linking structural features of protein complexes and biological function.

    PubMed

    Sowmya, Gopichandran; Breen, Edmond J; Ranganathan, Shoba

    2015-09-01

    Protein-protein interaction (PPI) establishes the central basis for complex cellular networks in a biological cell. Association of proteins with other proteins occurs at varying affinities, yet with a high degree of specificity. PPIs lead to diverse functionality such as catalysis, regulation, signaling, immunity, and inhibition, playing a crucial role in functional genomics. The molecular principle of such interactions is often elusive in nature. Therefore, a comprehensive analysis of known protein complexes from the Protein Data Bank (PDB) is essential for the characterization of structural interface features to determine structure-function relationship. Thus, we analyzed a nonredundant dataset of 278 heterodimer protein complexes, categorized into major functional classes, for distinguishing features. Interestingly, our analysis has identified five key features (interface area, interface polar residue abundance, hydrogen bonds, solvation free energy gain from interface formation, and binding energy) that are discriminatory among the functional classes using Kruskal-Wallis rank sum test. Significant correlations between these PPI interface features amongst functional categories are also documented. Salt bridges correlate with interface area in regulator-inhibitors (r = 0.75). These representative features have implications for the prediction of potential function of novel protein complexes. The results provide molecular insights for better understanding of PPIs and their relation to biological functions. PMID:26131659

  13. 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

  14. Optimized Affinity Capture of Yeast Protein Complexes.

    PubMed

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

    2016-01-01

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

  15. Effective Models for Electron Tansfer in Proteins - Connection Between Pathway and Detailed Hamiltonians

    NASA Astrophysics Data System (ADS)

    Balabin, I. A.; Onichic, J. N.

    1997-03-01

    Understanding how the protein molecular structure controls the electron transfer (ET) rate is critical for both achieving an insight into vital bioenergetic reactions and designing new ET proteins. We develop and test a new approach for computing ET tunneling matrix elements. Our goal is to provide quantitative results for large molecules with limited computer resources. This connection between simple models and more detailed atomistic models will also provide a better understanding of the basic features that control the ET mechanism. We introduce a series of simple Hamiltonians that incorporate effects of complex molecular structure on the ET rate. Electronic orbital interactions are categorized as classes, and only the most important of them are included. The remaining orbitals are incorporated by means of effective (dependent on the tunneling energy) interaction parameters. Calculations with these Hamiltonians are compared with ``exact'' extended Huckel-level results for several biological and chemically-designed systems. The suggested approach integrates quantum chemical and pathway-like methods. Quantitative calculations with limited computer resources and identification of the domains dominating ET are now in reach. This new developed approach integrates quantum chemistry and pathway-like methods.

  16. Bringing single-molecule spectroscopy to macromolecular protein complexes

    PubMed Central

    Joo, Chirlmin; Fareh, Mohamed; Kim, V. Narry

    2013-01-01

    Single-molecule fluorescence spectroscopy offers real-time, nanometer-resolution information. Over the past two decades, this emerging single-molecule technique has been rapidly adopted to investigate the structural dynamics and biological functions of proteins. Despite this remarkable achievement, single-molecule fluorescence techniques must be extended to macromolecular protein complexes that are physiologically more relevant for functional studies. In this review, we present recent major breakthroughs for investigating protein complexes within cell extracts using single-molecule fluorescence. We outline the challenges, future prospects and potential applications of these new single-molecule fluorescence techniques in biological and clinical research. PMID:23200186

  17. Electrophoresis of proteins and protein-protein complexes in native polyacrylamide gels using a horizontal gel apparatus.

    PubMed

    Su, C; Wang, F; Ciolek, D; Pan, Y C

    1994-11-15

    Electrophoresis of proteins and protein-protein complexes in polyacrylamide gels under native conditions using a horizontal gel apparatus is described. The advantage of this system is that it permits the detection of both negatively and positively charged proteins as well as protein-protein complexes in the same gel. During electrophoresis, a continuous gel sandwiched between two glass plates is placed horizontally on the platform and submerged in a reservoir buffer. The sample wells are made along the center of the gel, allowing positively and negatively charged proteins to migrate toward the cathode and anode, respectively. Several proteins with varying molecular weights and isoelectric point (pI) values and pairs of proteins capable of forming protein-protein complexes were chosen as model systems to illustrate the methodology. The effects of several parameters on the performance of the gel system including protein molecular weight, pI, and gel concentration were also examined and the results obtained by this method are comparable to those obtained by the vertical system. Following electrophoresis, both negatively and positively charged proteins as well as protein-protein complexes can be transferred by electroblotting onto polyvinylidene difluoride membranes for further analyses. PMID:7695108

  18. 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

  19. Capillary Isoelectric Focusing-Mass Spectrometry of Proteins and Protein Complexes

    SciTech Connect

    Martinovic, Suzana; Pasa-Tolic, Liljiana; Smith, Richard D.

    2004-10-01

    Complex proteome samples require efficient separation and detection methods in order to characterize their protein components. On-line combination of capillary isoelectric focusing (CIEF) with electrospray ionization (ESI) mass spectrometry (MS) is shown as an effective method to analyze complex protein mixtures. Our experience with several microorganisms allowed us to establish successful experimental protocol. Here we use the example of E. coli whole cell lysate for the CIEF separation and MS detection on the intact protein level. The protocol was further adapted for the analysis of the mixture of non-covalent complexes on the intact complex level.

  20. 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.

  1. Negative Ions Enhance Survival of Membrane Protein Complexes.

    PubMed

    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. Graphical Abstract ᅟ. PMID:27106602

  2. 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.

    2016-04-01

    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.

  3. A-Kinase Anchoring Proteins: From protein complexes to physiology and disease

    PubMed Central

    Carnegie, Graeme K.; Means, Christopher K.; Scott, John D.

    2009-01-01

    Protein scaffold complexes are a key mechanism by which a common signaling pathway can serve many different functions. Sequestering a signaling enzyme to a specific subcellular environment not only ensures that the enzyme is near its relevant targets, but also segregates this activity to prevent indiscriminate phosphorylation of other substrates. One family of diverse, well-studied scaffolding proteins are the A-kinase anchoring proteins (AKAPs). These anchoring proteins form multi-protein complexes that integrate cAMP signaling with other pathways and signaling events. In this review we focus on recent advances in the elucidation of AKAP function. PMID:19319965

  4. Molecular dynamics simulation strategies for protein-micelle complexes.

    PubMed

    Cheng, Xi; Kim, Jin-Kyoung; Kim, Yangmee; Bowie, James U; Im, Wonpil

    2016-07-01

    The structure and stability of membrane proteins can vary widely in different detergents and this variability has great practical consequences for working with membrane proteins. Nevertheless, the mechanisms that operate to alter the behavior of proteins in micelles are poorly understood and not predictable. Atomic simulations could provide considerable insight into these mechanisms. Building protein-micelle complexes for simulation is fraught with uncertainty, however, in part because it is often unknown how many detergent molecules are present in the complex. Here, we describe several convenient ways to employ Micelle Builder in CHARMM-GUI to rapidly construct protein-micelle complexes and performed simulations of the isolated voltage-sensor domain of voltage-dependent potassium-selective channel and an antimicrobial peptide papiliocin with varying numbers of detergents. We found that once the detergent number exceeds a threshold, protein-detergent interactions change very little and remain very consistent with experimental observations. Our results provide a platform for future studies of the interplays between protein structure and detergent properties at the atomic level. This article is part of a Special Issue entitled: Membrane Proteins edited by J.C. Gumbart and Sergei Noskov. PMID:26679426

  5. 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.

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

    PubMed

    Ren, Jun; Zhou, Wei; Wang, Jianxin

    2014-01-01

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

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

    PubMed Central

    Ren, Jun; Zhou, Wei; Wang, Jianxin

    2014-01-01

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

  8. 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

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

    PubMed

    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

  10. Detecting protein complexes in protein interaction networks using a ranking algorithm with a refined merging procedure

    PubMed Central

    2014-01-01

    Background Developing suitable methods for the identification of protein complexes remains an active research area. It is important since it allows better understanding of cellular functions as well as malfunctions and it consequently leads to producing more effective cures for diseases. In this context, various computational approaches were introduced to complement high-throughput experimental methods which typically involve large datasets, are expensive in terms of time and cost, and are usually subject to spurious interactions. Results In this paper, we propose ProRank+, a method which detects protein complexes in protein interaction networks. The presented approach is mainly based on a ranking algorithm which sorts proteins according to their importance in the interaction network, and a merging procedure which refines the detected complexes in terms of their protein members. ProRank + was compared to several state-of-the-art approaches in order to show its effectiveness. It was able to detect more protein complexes with higher quality scores. Conclusions The experimental results achieved by ProRank + show its ability to detect protein complexes in protein interaction networks. Eventually, the method could potentially identify previously-undiscovered protein complexes. The datasets and source codes are freely available for academic purposes at http://faculty.uaeu.ac.ae/nzaki/Research.htm. PMID:24944073

  11. 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.

  12. 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

  13. Rapid purification of protein complexes from mammalian cells

    PubMed Central

    Medina, Dan; Moskowitz, Neal; Khan, Subarna; Christopher, Scott; Germino, Joseph

    2000-01-01

    The evaluation of the protein binding partner(s) of biologically important proteins is currently an area of intense research, especially since the development of the yeast two-hybrid assay. However, not all protein–protein interactions uncovered by this assay are biologically relevant and another confirmatory assay must be performed. Ideally, this assay should be rapid, versatile and performed under conditions which mimic the ‘normal’ physiological state as closely as possible. Towards this goal, we have constructed two eukaryotic expression vectors that facilitate the purification of a protein of interest, along with any associated proteins, from mammalian cells. These vectors incorporate the following features: (i) a tetracycline-responsive promoter so that the level of protein production can be regulated; (ii) an N-terminal glutathione S-transferase tag or a triple repeat of the HA1 epitope, to facilitate purification of the protein either by glutathione affinity chromatography or immunoprecipitation, respectively, followed by a multiple cloning site; (iii) the gene for the enhanced green fluorescent protein (for detection of the presence of the fusion protein and subcellular localization); (iv) a puromycin marker for the selection of stable transformants; (v) a truncated EBNA protein and oriP sequence for episomal replication of the vector. These latter two features permit expansion of small cultures of transfected cells under puromycin selection, thereby increasing the amount of tagged protein that can be purified. We show that these vectors can be used to direct the doxycycline-inducible expresssion of tagged proteins and to recover tagged CIP1–p21 protein complexes from HeLa cells. Furthermore, we show that these tagged p21-purified complexes contain both cyclin A and Cdk2, which are known to interact with p21, but not β-actin. PMID:10871384

  14. Functional connectivity between posterior hippocampus and retrosplenial complex predicts individual differences in navigational ability.

    PubMed

    Sulpizio, Valentina; Boccia, Maddalena; Guariglia, Cecilia; Galati, Gaspare

    2016-07-01

    Individuals vary widely in their ability to orient and navigate within the environment. Previous neuroimaging research has shown that hippocampus (HC) and scene-responsive regions (retrosplenial complex [RSC] and parahippocampal gyrus/parahippocampal place area [PPA]) were crucial for spatial orienting and navigation. Resting-state functional connectivity and a self-reported questionnaire of navigational ability were used to examine the hypothesis that the pattern of reciprocal connections between these regions reflects individual differences in spatial navigation. It was found that the functional connectivity between the posterior HC and RSC was significantly higher in good than in poor navigators. These results confirmed the crucial role of hippocampal and extra-hippocampal regions in spatial navigation and provided new insight into how spontaneous brain activity may account for individual differences in spatial ability. © 2016 Wiley Periodicals, Inc. PMID:27013151

  15. CSF proteins and resting-state functional connectivity in Parkinson disease

    PubMed Central

    Koller, Jonathan M.; Snyder, Abraham Z.; Buddhala, Chandana; Kotzbauer, Paul T.; Perlmutter, Joel S.

    2015-01-01

    Objective: The purpose of this study was to investigate the relationship between disruption of MRI-measured resting-state functional connectivity (rs-fcMRI) brain networks and CSF levels of potentially pathogenic proteins that reflect brain pathology in Parkinson disease (PD). Methods: PD participants without dementia (n = 43) and age-matched controls (n = 22) had lumbar punctures to measure CSF protein levels, Pittsburgh compound B (PiB)–PET imaging, and rs-fcMRI while off medication. Imaging analyses focused on 5 major resting-state networks as well as the striatum. Results: Participants with PD had significantly reduced sensorimotor functional connectivity, which correlated with reduced CSF levels of α-synuclein. The PD group also had significantly stronger default mode network functional connectivity that did not correlate with CSF β-amyloid (Aβ)42 or PiB uptake. In contrast, default mode network functional connectivity in the control group did correlate with CSF Aβ42 levels. Functional connectivity was similar between groups in the dorsal attention, control, and salience networks. Conclusion: These results suggest that abnormal α-synuclein accumulation, but not Aβ, contributes to the disruption of motor-related functional connectivity in PD. Furthermore, correlating CSF protein measures with the strength of resting-state networks provides a direct link between abnormal α-synuclein metabolism and disrupted brain function in PD. PMID:25979701

  16. Analyzing Large Protein Complexes by Structural Mass Spectrometry

    PubMed Central

    Kirshenbaum, Noam; Michaelevski, Izhak; Sharon, Michal

    2010-01-01

    Living cells control and regulate their biological processes through the coordinated action of a large number of proteins that assemble themselves into an array of dynamic, multi-protein complexes1. To gain a mechanistic understanding of the various cellular processes, it is crucial to determine the structure of such protein complexes, and reveal how their structural organization dictates their function. Many aspects of multi-protein complexes are, however, difficult to characterize, due to their heterogeneous nature, asymmetric structure, and dynamics. Therefore, new approaches are required for the study of the tertiary levels of protein organization. One of the emerging structural biology tools for analyzing macromolecular complexes is mass spectrometry (MS)2-5. This method yields information on the complex protein composition, subunit stoichiometry, and structural topology. The power of MS derives from its high sensitivity and, as a consequence, low sample requirement, which enables examination of protein complexes expressed at endogenous levels. Another advantage is the speed of analysis, which allows monitoring of reactions in real time. Moreover, the technique can simultaneously measure the characteristics of separate populations co-existing in a mixture. Here, we describe a detailed protocol for the application of structural MS to the analysis of large protein assemblies. The procedure begins with the preparation of gold-coated capillaries for nanoflow electrospray ionization (nESI). It then continues with sample preparation, emphasizing the buffer conditions which should be compatible with nESI on the one hand, and enable to maintain complexes intact on the other. We then explain, step-by-step, how to optimize the experimental conditions for high mass measurements and acquire MS and tandem MS spectra. Finally, we chart the data processing and analyses that follow. Rather than attempting to characterize every aspect of protein assemblies, this protocol

  17. 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

  18. Connecting protein and mRNA burst distributions for stochastic models of gene expression

    NASA Astrophysics Data System (ADS)

    Elgart, Vlad; Jia, Tao; Fenley, Andrew T.; Kulkarni, Rahul

    2011-08-01

    The intrinsic stochasticity of gene expression can lead to large variability in protein levels for genetically identical cells. Such variability in protein levels can arise from infrequent synthesis of mRNAs which in turn give rise to bursts of protein expression. Protein expression occurring in bursts has indeed been observed experimentally and recent studies have also found evidence for transcriptional bursting, i.e. production of mRNAs in bursts. Given that there are distinct experimental techniques for quantifying the noise at different stages of gene expression, it is of interest to derive analytical results connecting experimental observations at different levels. In this work, we consider stochastic models of gene expression for which mRNA and protein production occurs in independent bursts. For such models, we derive analytical expressions connecting protein and mRNA burst distributions which show how the functional form of the mRNA burst distribution can be inferred from the protein burst distribution. Additionally, if gene expression is repressed such that observed protein bursts arise only from single mRNAs, we show how observations of protein burst distributions (repressed and unrepressed) can be used to completely determine the mRNA burst distribution. Assuming independent contributions from individual bursts, we derive analytical expressions connecting means and variances for burst and steady-state protein distributions. Finally, we validate our general analytical results by considering a specific reaction scheme involving regulation of protein bursts by small RNAs. For a range of parameters, we derive analytical expressions for regulated protein distributions that are validated using stochastic simulations. The analytical results obtained in this work can thus serve as useful inputs for a broad range of studies focusing on stochasticity in gene expression.

  19. 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.

  20. Towards the development of Bacillus subtilis as a cell factory for membrane proteins and protein complexes

    PubMed Central

    Zweers, Jessica C; Barák, Imrich; Becher, Dörte; Driessen, Arnold JM; Hecker, Michael; Kontinen, Vesa P; Saller, Manfred J; Vavrová, L'udmila; van Dijl, Jan Maarten

    2008-01-01

    Background The Gram-positive bacterium Bacillus subtilis is an important producer of high quality industrial enzymes and a few eukaryotic proteins. Most of these proteins are secreted into the growth medium, but successful examples of cytoplasmic protein production are also known. Therefore, one may anticipate that the high protein production potential of B. subtilis can be exploited for protein complexes and membrane proteins to facilitate their functional and structural analysis. The high quality of proteins produced with B. subtilis results from the action of cellular quality control systems that efficiently remove misfolded or incompletely synthesized proteins. Paradoxically, cellular quality control systems also represent bottlenecks for the production of various heterologous proteins at significant concentrations. Conclusion While inactivation of quality control systems has the potential to improve protein production yields, this could be achieved at the expense of product quality. Mechanisms underlying degradation of secretory proteins are nowadays well understood and often controllable. It will therefore be a major challenge for future research to identify and modulate quality control systems of B. subtilis that limit the production of high quality protein complexes and membrane proteins, and to enhance those systems that facilitate assembly of these proteins. PMID:18394159

  1. A secretory kinase complex regulates extracellular protein phosphorylation.

    PubMed

    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. PMID:25789606

  2. 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

  3. Displacement affinity chromatography of protein phosphatase one (PP1) complexes

    PubMed Central

    Moorhead, Greg BG; Trinkle-Mulcahy, Laura; Nimick, Mhairi; De Wever, Veerle; Campbell, David G; Gourlay, Robert; Lam, Yun Wah; Lamond, Angus I

    2008-01-01

    Background Protein phosphatase one (PP1) is a ubiquitously expressed, highly conserved protein phosphatase that dephosphorylates target protein serine and threonine residues. PP1 is localized to its site of action by interacting with targeting or regulatory proteins, a majority of which contains a primary docking site referred to as the RVXF/W motif. Results We demonstrate that a peptide based on the RVXF/W motif can effectively displace PP1 bound proteins from PP1 retained on the phosphatase affinity matrix microcystin-Sepharose. Subsequent co-immunoprecipitation experiments confirmed that each identified binding protein was either a direct PP1 interactor or was in a complex that contains PP1. Our results have linked PP1 to numerous new nuclear functions and proteins, including Ki-67, Rif-1, topoisomerase IIα, several nuclear helicases, NUP153 and the TRRAP complex. Conclusion This modification of the microcystin-Sepharose technique offers an effective means of purifying novel PP1 regulatory subunits and associated proteins and provides a simple method to uncover a link between PP1 and additional cellular processes. PMID:19000314

  4. 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

  5. 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.

  6. 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

  7. 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.

  8. Deciphering preferential interactions within supramolecular protein complexes: the proteasome case

    PubMed Central

    Fabre, Bertrand; Lambour, Thomas; Garrigues, Luc; Amalric, François; Vigneron, Nathalie; Menneteau, Thomas; Stella, Alexandre; Monsarrat, Bernard; Van den Eynde, Benoît; Burlet-Schiltz, Odile; Bousquet-Dubouch, Marie-Pierre

    2015-01-01

    In eukaryotic cells, intracellular protein breakdown is mainly performed by the ubiquitin–proteasome system. Proteasomes are supramolecular protein complexes formed by the association of multiple sub-complexes and interacting proteins. Therefore, they exhibit a very high heterogeneity whose function is still not well understood. Here, using a newly developed method based on the combination of affinity purification and protein correlation profiling associated with high-resolution mass spectrometry, we comprehensively characterized proteasome heterogeneity and identified previously unknown preferential associations within proteasome sub-complexes. In particular, we showed for the first time that the two main proteasome subtypes, standard proteasome and immunoproteasome, interact with a different subset of important regulators. This trend was observed in very diverse human cell types and was confirmed by changing the relative proportions of both 20S proteasome forms using interferon-γ. The new method developed here constitutes an innovative and powerful strategy that could be broadly applied for unraveling the dynamic and heterogeneous nature of other biologically relevant supramolecular protein complexes. PMID:25561571

  9. Architecture and function of IFT complex proteins in ciliogenesis

    PubMed Central

    Taschner, Michael; Bhogaraju, Sagar; Lorentzen, Esben

    2014-01-01

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

    PubMed

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

    2010-01-01

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

  11. 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

  12. 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

  13. 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

  14. Isotopically coded cleavable cross-linker for studying protein-protein interaction and protein complexes.

    PubMed

    Petrotchenko, Evgeniy V; Olkhovik, Vyacheslav K; Borchers, Christoph H

    2005-08-01

    An emerging approach for studying protein-protein interaction in complexes is the combination of chemical cross-linking and mass spectrometric analysis of the cross-linked peptides (cross-links) obtained after proteolysis of the complex. This approach, however, has several challenges and limitations, including the difficulty of detecting the cross-links, the potential interference from non-informative "cross-linked peptides" (dead end and intrapeptide cross-links), and unambiguous identification of the cross-links by mass spectrometry. Thus, we have synthesized an isotopically coded ethylene glycol bis(succinimidylsuccinate) derivate (D12-EGS), which contains 12 deuterium atoms for easy detection of cross-links when applied in a 1:1 mixture with its H12 counterpart and is also cleavable for releasing the cross-linked peptides allowing unambiguous identification by MS sequencing. Moreover, hydrolytic cleavage permits rapid distinguishing between different types of cross-links. Cleavage of a dead end cross-link produces a doublet with peaks 4.03 Da apart, with the lower peak appearing at a molecular mass 162 Da lower than the mass of the H12 form of the original cross-linked peptide. Cleavage of an intrapeptide cross-link leads to a doublet 8.05 Da apart and 62 Da lower than the molecular mass of the H12 form of the original cross-linked peptide. Cleavage of an interpeptide cross-link forms a pair of 4.03-Da doublets, with the lower mass member of each pair each shifted up from its unmodified molecular weight by 82 Da because of the attached portion of the cross-linker. All of this information has been incorporated into a software algorithm allowing automatic screening and detection of cross-links and cross-link types in matrix-assisted laser desorption/ionization mass spectra. In summary, the ease of detection of these species through the use of an isotopically coded cleavable cross-linker and our software algorithm, followed by mass spectrometric sequencing of the

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

    PubMed

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

    2015-01-01

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

  16. Protein corona – from molecular adsorption to physiological complexity

    PubMed Central

    Docter, Dominic; Maskos, Michael

    2015-01-01

    Summary 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. PMID:25977856

  17. Protein Ligand Complex Guided Approach for Virtual Screening.

    PubMed

    Karthikeyan, Muthukumarasamy; Pandit, Deepak; Vyas, Renu

    2015-01-01

    The target ligand association data is a rich source of information which is not exploited enough for drug design efforts in virtual screening. A java based open-source toolkit for Protein Ligand Network Extraction (J-ProLiNE) focused on protein-ligand complex analysis with several features integrated in a distributed computing network has been developed. Sequence alignment and similarity search components have been automated to yield local, global alignment scores along with similarity and distance scores. 10000 proteins with co-crystallized ligands from pdb and MOAD databases were extracted and analyzed for revealing relationships between targets, ligands and scaffolds. Through this analysis, we could generate a protein ligand network to identify the promiscuous and selective scaffolds for multiple classes of proteins targets. Using J-ProLiNE we created a 507 x 507 matrix of protein targets and native ligands belonging to six enzyme classes and analyzed the results to elucidate the protein-protein, protein-ligand and ligand-ligand interactions. In yet another application of the J-ProLiNE software, we were able to process kinase related information stored in US patents to construct disease-gene-ligand-scaffold networks. It is hoped that the studies presented here will enable target ligand knowledge based virtual screening for inhibitor design. PMID:26138572

  18. Pigment Analysis of Chloroplast Pigment-Protein Complexes in Wheat

    PubMed Central

    Eskins, Kenneth; Duysen, Murray E.; Olson, Linda

    1983-01-01

    Pigment-protein complexes separated from wheat (Triticum aestivum L. selection ND96-25 by two gel electrophoresis techniques were analyzed by high-performance liquid chromatography for chlorophylls and carotenoids. The two techniques are compared, and pigment analyses are given for the major reaction centers and light-harvesting complexes. Reaction centers contain mostly chlorophyll a, carotene, and lutein, whereas light-harvesting complexes contain chlorophyll a, chlorophyll b, lutein, and neoxanthin. The amounts of violaxanthin are variable. Images Fig. 1 PMID:16662906

  19. Cardiac mitochondrial matrix and respiratory complex protein phosphorylation

    PubMed Central

    Covian, Raul

    2012-01-01

    It has become appreciated over the last several years that protein phosphorylation within the cardiac mitochondrial matrix and respiratory complexes is extensive. Given the importance of oxidative phosphorylation and the balance of energy metabolism in the heart, the potential regulatory effect of these classical signaling events on mitochondrial function is of interest. However, the functional impact of protein phosphorylation and the kinase/phosphatase system responsible for it are relatively unknown. Exceptions include the well-characterized pyruvate dehydrogenase and branched chain α-ketoacid dehydrogenase regulatory system. The first task of this review is to update the current status of protein phosphorylation detection primarily in the matrix and evaluate evidence linking these events with enzymatic function or protein processing. To manage the scope of this effort, we have focused on the pathways involved in energy metabolism. The high sensitivity of modern methods of detecting protein phosphorylation and the low specificity of many kinases suggests that detection of protein phosphorylation sites without information on the mole fraction of phosphorylation is difficult to interpret, especially in metabolic enzymes, and is likely irrelevant to function. However, several systems including protein translocation, adenine nucleotide translocase, cytochrome c, and complex IV protein phosphorylation have been well correlated with enzymatic function along with the classical dehydrogenase systems. The second task is to review the current understanding of the kinase/phosphatase system within the matrix. Though it is clear that protein phosphorylation occurs within the matrix, based on 32P incorporation and quantitative mass spectrometry measures, the kinase/phosphatase system responsible for this process is ill-defined. An argument is presented that remnants of the much more labile bacterial protein phosphoryl transfer system may be present in the matrix and that the

  20. 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

  1. 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…

  2. 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

  3. 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

  4. 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

  5. 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

  6. Structural Reconstruction of Protein-Protein Complexes Involved in Intracellular Signaling.

    PubMed

    Kirsch, Klára; Sok, Péter; Reményi, Attila

    2016-01-01

    Signaling complexes within the cell convert extracellular cues into physiological outcomes. Their assembly involves signaling enzymes, allosteric regulators and scaffold proteins that often contain long stretches of disordered protein regions, display multi-domain architectures, and binding affinity between individual components is low. These features are indispensable for their central roles as dynamic information processing hubs, on the other hand they also make reconstruction of structurally homogeneous complex samples highly challenging. In this present chapter we discuss protein machinery which influences extracellular signal reception, intracellular pathway activity, and cytoskeletal or transcriptional activity. PMID:27165334

  7. Self-assembly of DNA double-double crossover complexes into high-density, doubly connected, planar structures.

    PubMed

    Reishus, Dustin; Shaw, Bilal; Brun, Yuriy; Chelyapov, Nickolas; Adleman, Leonard

    2005-12-21

    We designed a molecular complex, the double-double crossover, consisting of four DNA double helices connected by six reciprocal exchanges. Atomic force micrographs suggest that double-double crossover complexes self-assemble into high-density, doubly connected, two-dimensional, planar structures. Such structures may be suitable as substrates for the deposition of nanomaterials in the creation of high-density electrical and quantum devices. We speculate about a modified double-double crossover complex that might self-assemble into high-density, doubly connected, three-dimensional structures. PMID:16351073

  8. Modeling of protein binary complexes using structural mass spectrometry data

    PubMed Central

    Kamal, J.K. Amisha; Chance, Mark R.

    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. PMID:18042684

  9. From Nonspecific DNA–Protein Encounter Complexes to the Prediction of DNA–Protein Interactions

    PubMed Central

    Gao, Mu; Skolnick, Jeffrey

    2009-01-01

    DNA–protein interactions are involved in many essential biological activities. Because there is no simple mapping code between DNA base pairs and protein amino acids, the prediction of DNA–protein interactions is a challenging problem. Here, we present a novel computational approach for predicting DNA-binding protein residues and DNA–protein interaction modes without knowing its specific DNA target sequence. Given the structure of a DNA-binding protein, the method first generates an ensemble of complex structures obtained by rigid-body docking with a nonspecific canonical B-DNA. Representative models are subsequently selected through clustering and ranking by their DNA–protein interfacial energy. Analysis of these encounter complex models suggests that the recognition sites for specific DNA binding are usually favorable interaction sites for the nonspecific DNA probe and that nonspecific DNA–protein interaction modes exhibit some similarity to specific DNA–protein binding modes. Although the method requires as input the knowledge that the protein binds DNA, in benchmark tests, it achieves better performance in identifying DNA-binding sites than three previously established methods, which are based on sophisticated machine-learning techniques. We further apply our method to protein structures predicted through modeling and demonstrate that our method performs satisfactorily on protein models whose root-mean-square Cα deviation from native is up to 5 Å from their native structures. This study provides valuable structural insights into how a specific DNA-binding protein interacts with a nonspecific DNA sequence. The similarity between the specific DNA–protein interaction mode and nonspecific interaction modes may reflect an important sampling step in search of its specific DNA targets by a DNA-binding protein. PMID:19343221

  10. Two Clathrin Adaptor Protein Complexes Instruct Axon-Dendrite Polarity.

    PubMed

    Li, Pengpeng; Merrill, Sean A; Jorgensen, Erik M; Shen, Kang

    2016-05-01

    The cardinal feature of neuronal polarization is the establishment and maintenance of axons and dendrites. How axonal and dendritic proteins are sorted and targeted to different compartments is poorly understood. Here, we identified distinct dileucine motifs that are necessary and sufficient to target transmembrane proteins to either the axon or the dendrite through direct interactions with the clathrin-associated adaptor protein complexes (APs) in C. elegans. Axonal targeting requires AP-3, while dendritic targeting is mediated by AP-1. The axonal dileucine motif binds to AP-3 with higher efficiency than to AP-1. Both AP-3 and AP-1 are localized to the Golgi but occupy adjacent domains. We propose that AP-3 and AP-1 directly select transmembrane proteins and target them to axon and dendrite, respectively, by sorting them into distinct vesicle pools. PMID:27151641

  11. Over-producing soluble protein complex and validating protein-protein interaction through a new bacterial co-expression system.

    PubMed

    Zeng, Jumei; Zhang, Lei; Li, Yuqing; Wang, Yi; Wang, Mingchao; Duan, Xin; He, Zheng-Guo

    2010-01-01

    Many proteins exert their functions through a protein complex and protein-protein interactions. However, the study of these types of interactions is complicated when dealing with toxic or hydrophobic proteins. It is difficult to use the popular Escherichia coli host for their expression, as these proteins in all likelihood require a critical partner protein to ensure their proper folding and stability. In the present study, we have developed a novel co-expression vector, pHEX, which is compatible with, and thus can be partnered with, many commercially available E. coli vectors, such as pET, pGEX and pMAL. The pHEX contains the p15A origin of replication and a T7 promoter, which can over-produce a His-tagged recombinant protein. The new co-expression system was demonstrated to efficiently co-produce and co-purify heterodimeric protein complexes, for example PE25/PPE41 (Rv2430c/Rv2431c) and ESAT6/CFP10 (Rv3874/Rv3875), from the human pathogen Mycobacterium tuberculosis H37Rv. Furthermore, the system was also effectively used to characterize protein-protein interactions through convenient affinity tags. Using an in vivo pull-down assay, for the first time we have confirmed the presence of three pairs of PE/PPE-related novel protein interactions in this pathogen. In summary, a convenient and efficient co-expression vector system has been successfully developed. The new system should be applicable to any protein complex or any protein-protein interaction of interest in a wide range of biological organisms. PMID:19747546

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

    PubMed Central

    Yetnikoff, Leora; Lavezzi, Heather N.; Reichard, Rhett A.; Zahm, Daniel S.

    2014-01-01

    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 masssive 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 human. 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

  13. 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

  14. Molecular Signatures of Membrane Protein Complexes Underlying Muscular Dystrophy.

    PubMed

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

    2016-06-01

    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

  15. 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

  16. Plasmid-protein relaxation complexes in Staphylococcus aureus.

    PubMed

    Novick, R

    1976-09-01

    Protein-deoxyribonucleic acid relaxation complexes have been demonstrated for six Staphylococcus aureus plasmids out of sixteen examined. Four of these encode stretomycin resistence, have molecular weights of about 2.7 x 10(6), and are isolated as supercoiled molecules that are virtally 100% relaxable by treatment with sodium dodecyl sulfate. It is probable that these four isolates represent a single widely disseminated plasmid species. The other two plasmids showing relaxation complexes have molecular weights of about 3 x 10(6) and encode chloramphenicol resistance. The complexes in these cases are unstable, and it has not been possible to induce more than 50% relaxation by any of the standard treatments. Ten other plasmids do not show detectable complexes. These include three penicillinase plasmids, four tetracycline-resistance plasmids, one plasmid carrying kanamycin-neomycin resistance, and finally, two chloramphenicol-resistance plasmids. PMID:956124

  17. 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

  18. Chemiluminescence enzyme immunoassay using ProteinA-bacterial magnetite complex

    NASA Astrophysics Data System (ADS)

    Matsunaga, Tadashi; Sato, Rika; Kamiya, Shinji; Tanaka, Tsuyosi; Takeyama, Haruko

    1999-04-01

    Bacterial magnetic particles (BMPs) which have ProteinA expressed on their surface were constructed using magA which is a key gene in BMP biosynthesis in the magnetic bacterium Magnetospirillum sp. AMB-1. Homogenous chemiluminescence enzyme immunoassay using antibody bound ProteinA-BMP complexes was developed for detection of human IgG. A good correlation between the luminescence yield and the concentration of human IgG was obtained in the range of 1-10 3 ng/ml.

  19. 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

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

    PubMed

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

    2014-01-01

    The analyses of protein-protein interactions are 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

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

    PubMed

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

    2016-06-01

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

  2. Analysis of secondary structural and physicochemical changes in protein-protein complexes.

    PubMed

    Saranya, N; Saravanan, K M; Michael Gromiha, M; Selvaraj, S

    2016-03-01

    Conformation switching in protein-protein complexes is considered important for the molecular recognition process. Overall analysis of 123 protein-protein complexes in a benchmark data-set showed that 6.8% of residues switched over their secondary structure conformation upon complex formation. Amino acid residue-wise preference for conformation change has been analyzed in binding and non-binding site residues separately. In this analysis, residues such as Ser, Leu, Glu, and Lys had higher frequency of secondary structural conformation change. The change of helix to coil and sheet to coil conformation and vice versa has been observed frequently, whereas the conformation change of helix to extended sheet occurred rarely in the studied complexes. Influence of conformation change toward the N and C terminal on either side of the binding site residues has been analyzed. Further, analysis on φ and ψ angle variation, conservation, stability, and solvent accessibility have been performed on binding site residues. Knowledge obtained from the present study could be effectively employed in the protein-protein modeling and docking studies. PMID:25990569

  3. Complex architecture of major histocompatibility complex class II promoters: reiterated motifs and conserved protein-protein interactions.

    PubMed Central

    Jabrane-Ferrat, N; Fontes, J D; Boss, J M; Peterlin, B M

    1996-01-01

    The S box (also known as at the H, W, or Z box) is the 5'-most element of the conserved upstream sequences in promoters of major histocompatibility complex class II genes. It is important for their B-cell-specific and interferon gamma-inducible expression. In this study, we demonstrate that the S box represents a duplication of the downstream X box. First, RFX, which is composed of the RFX5-p36 heterodimer that binds to the X box, also binds to the S box and its 5'-flanking sequence. Second, NF-Y, which binds to the Y box and increases interactions between RFX and the X box, also increases the binding of RFX to the S box. Third, RFXs bound to S and X boxes interact with each other in a spatially constrained manner. Finally, we confirmed these protein-protein and protein-DNA interactions by expressing a hybrid RFX5-VP16 protein in cells. We conclude that RFX binds to S and X boxes and that complex interactions between RFX and NF-Y direct B-cell-specific and interferon gamma-inducible expression or major histocompatibility complex class II genes. PMID:8756625

  4. Size dependent complexity of sequences in protein families

    NASA Astrophysics Data System (ADS)

    Li, J.; Wang, J.; Wang, W.

    2005-10-01

    The size dependent complexity of protein sequences in various families in the FSSP database is characterized by sequence entropy, sequence similarity and sequence identity. As the average length Lf of sequences in the family increases, an increasing trend of the sequence entropy and a decreasing trend of the sequence similarity and sequence identity are found. As Lf increases beyond 250, a saturation of the sequence entropy, the sequence similarity and the sequence identity is observed. Such a saturated behavior of complexity is attributed to the saturation of the probability Pg of global (long-range) interactions in protein structures when Lf >250. It is also found that the alphabet size of residue types describing the sequence diversity depends on the value of Lf, and becomes saturated at 12.

  5. Protein-protein complex structure predictions by multimeric threading and template recombination

    PubMed Central

    Mukherjee, Srayanta; Zhang, Yang

    2011-01-01

    Summary The number of protein-protein complex structures is nearly 6-times smaller than that of tertiary structures in PDB which limits the power of homology-based approaches to complex structure modeling. We present a new threading-recombination approach, COTH, to boost the protein complex structure library by combining tertiary structure templates with complex alignments. The query sequences are first aligned to complex templates using a modified dynamic programming algorithm, guided by ab initio binding-site predictions. The monomer alignments are then shifted to the multimeric template framework by structural alignments. COTH was tested on 500 non-homologous dimeric proteins, which can successfully detect correct templates for half of the cases after homologous templates are excluded, which significantly outperforms conventional homology modeling algorithms. It also shows a higher accuracy in interface modeling than rigid-body docking of unbound structures from ZDOCK although with lower coverage. These data demonstrate new avenues to model complex structures from non-homologous templates. PMID:21742262

  6. The small G-protein MglA connects to the MreB actin cytoskeleton at bacterial focal adhesions.

    PubMed

    Treuner-Lange, Anke; Macia, Eric; Guzzo, Mathilde; Hot, Edina; Faure, Laura M; Jakobczak, Beata; Espinosa, Leon; Alcor, Damien; Ducret, Adrien; Keilberg, Daniela; Castaing, Jean Philippe; Lacas Gervais, Sandra; Franco, Michel; Søgaard-Andersen, Lotte; Mignot, Tâm

    2015-07-20

    In Myxococcus xanthus the gliding motility machinery is assembled at the leading cell pole to form focal adhesions, translocated rearward to propel the cell, and disassembled at the lagging pole. We show that MglA, a Ras-like small G-protein, is an integral part of this machinery. In this function, MglA stimulates the assembly of the motility complex by directly connecting it to the MreB actin cytoskeleton. Because the nucleotide state of MglA is regulated spatially and MglA only binds MreB in the guanosine triphosphate-bound form, the motility complexes are assembled at the leading pole and dispersed at the lagging pole where the guanosine triphosphatase activating protein MglB disrupts the MglA-MreB interaction. Thus, MglA acts as a nucleotide-dependent molecular switch to regulate the motility machinery spatially. The function of MreB in motility is independent of its function in peptidoglycan synthesis, representing a coopted function. Our findings highlight a new function for the MreB cytoskeleton and suggest that G-protein-cytoskeleton interactions are a universally conserved feature. PMID:26169353

  7. 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.

  8. 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

  9. 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. PMID:26971075

  10. 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

  11. 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. PMID:27448142

  12. Enhanced ad hoc wireless connectivity in complex environment using small radio repeater systems

    NASA Astrophysics Data System (ADS)

    Sarabandi, Kamal; Song, Young Jun; Oh, Jungsuek

    2011-06-01

    Ad hoc communication among small robotic platforms in complex indoor environment is further challenged by three limiting factors: 1) limited power, 2) small size antennas, and 3) near-ground operation. In complex environments such as indoor scenarios often times the line-of-sight communication cannot be established and the wireless connectivity must rely on multi-path propagation. As a result, the propagation path-loss is much higher than free-space, and more power will be needed to obtain the need coverage. Near ground operation also leads to increased path-loss. To maintain the network connectivity without increasing the required power a novel high gain miniaturized radio repeater is presented. Unlike existing repeater systems, this system utilizes two closely spaced low profile miniaturized planar antennas capable of producing omnidirectional and vertical radiation patterns as well as a channel isolator layer that serves to decouple the adjacent antennas. The meta-material based channel isolator serves as an electromagnetic shield, thus enabling it to be built in a sub-wavelength size of 0.07λ0 2 × λ0/70, the smallest repeater ever built. Also wave propagation simulations have been conducted to determine the required gain of such repeaters so to ensure the signal from the repeater is the dominant component. A prototype of the small radio repeater is fabricated to verify the design performance through a standard free-space measurement setup.

  13. 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

  14. 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.

  15. 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

  16. 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

  17. Radioprotection by polyethylene glycol-protein complexes in mice

    SciTech Connect

    Gray, B.H.; Stull, R.W.

    1983-03-01

    Polyethylene glycol of about 5000 D was activated with cyanuric chloride, and the activated compound was complexed to each of three proteins. Polyethylene glycol-superoxide dismutase and polyethylene glycol-catalase were each radioprotectants when administered prophylactically to female B6CBF1 mice before irradiation. The dose reduction factor for these mice was 1.2 when 5000 units of polyethylene glycol-catalase was administered before /sup 60/Co irradiation. Female B6CBF1 mice administered prophylactic intravenous injections of catalase, polyethylene glycol-albumin, or heat-denatured polyethylene glycol-catalase had survival rates similar to phosphate-buffered saline-injected control mice following /sup 60/Co irradiation. Polyethylene glycol-superoxide dismutase and polyethylene glycol-catalase have radioprotective activity in B6CBF1 mice, which appears to depend in part on enzymatic activities of the complex. However, no radioprotective effect was observed in male C57BL/6 mice injected with each polyethylene glycol-protein complex at either 3 or 24 hr before irradiation. The mechanism for radioprotection by these complexes may depend in part on other factors.

  18. Taking advantage of local structure descriptors to analyze interresidue contacts in protein structures and protein complexes.

    PubMed

    Martin, Juliette; Regad, Leslie; Etchebest, Catherine; Camproux, Anne-Claude

    2008-11-15

    Interresidue protein contacts in proteins structures and at protein-protein interface are classically described by the amino acid types of interacting residues and the local structural context of the contact, if any, is described using secondary structures. In this study, we present an alternate analysis of interresidue contact using local structures defined by the structural alphabet introduced by Camproux et al. This structural alphabet allows to describe a 3D structure as a sequence of prototype fragments called structural letters, of 27 different types. Each residue can then be assigned to a particular local structure, even in loop regions. The analysis of interresidue contacts within protein structures defined using Voronoï tessellations reveals that pairwise contact specificity is greater in terms of structural letters than amino acids. Using a simple heuristic based on specificity score comparison, we find that 74% of the long-range contacts within protein structures are better described using structural letters than amino acid types. The investigation is extended to a set of protein-protein complexes, showing that the similar global rules apply as for intraprotein contacts, with 64% of the interprotein contacts best described by local structures. We then present an evaluation of pairing functions integrating structural letters to decoy scoring and show that some complexes could benefit from the use of structural letter-based pairing functions. PMID:18491388

  19. mTOR Complexes Repress Hypertrophic Agonist-Stimulated Expression of Connective Tissue Growth Factor in Adult Cardiac Muscle Cells.

    PubMed

    Sundararaj, Kamala; Pleasant, Dorea L; Moschella, Phillip C; Panneerselvam, Kavin; Balasubramanian, Sundaravadivel; Kuppuswamy, Dhandapani

    2016-02-01

    Connective tissue growth factor (CTGF) is a fibrogenic cytokine that promotes fibrosis in various organs. In the heart, both cardiomyocytes (CM) and cardiac fibroblasts have been reported as a source of CTGF expression, aiding cardiac fibrosis. Although the mammalian target of rapamycin (mTOR) forms 2 distinct complexes, mTORC1 and mTORC2, and plays a central role in integrating biochemical signals for protein synthesis and cellular homeostasis, we explored its role in CTGF expression in adult feline CM. CM were stimulated with 10 μM phenylephrine (PE), 200 nM angiotensin (Ang), or 100 nM insulin for 24 hours. PE and Ang, but not insulin, caused an increase in CTGF mRNA expression with the highest expression observed with PE. Inhibition of mTOR with torin1 but not rapamycin significantly enhanced PE-stimulated CTGF expression. Furthermore, silencing of raptor and rictor using shRNA adenoviral vectors to suppress mTORC1 and mTORC2, respectively, or blocking phosphatidylinositol 3-kinase (PI3K) signaling with LY294002 (LY) or Akt signaling by dominant-negative Akt expression caused a substantial increase in PE-stimulated CTGF expression as measured by both mRNA and secreted protein levels. However, studies with dominant-negative delta isoform of protein kinase C demonstrate that delta isoform of protein kinase C is required for both agonist-induced CTGF expression and mTORC2/Akt-mediated CTGF suppression. Finally, PE-stimulated CTGF expression was accompanied with a corresponding increase in Smad3 phosphorylation and pretreatment of cells with SIS3, a Smad3 specific inhibitor, partially blocked the PE-stimulated CTGF expression. Therefore, a PI3K/mTOR/Akt axis plays a suppressive role on agonist-stimulated CTGF expression where the loss of this mechanism could be a contributing factor for the onset of cardiac fibrosis in the hypertrophying myocardium. PMID:26371948

  20. 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

  1. pyDockSAXS: protein-protein complex structure by SAXS and computational docking.

    PubMed

    Jiménez-García, Brian; Pons, Carles; Svergun, Dmitri I; Bernadó, Pau; Fernández-Recio, Juan

    2015-07-01

    Structural characterization of protein-protein interactions at molecular level is essential to understand biological processes and identify new therapeutic opportunities. However, atomic resolution structural techniques cannot keep pace with current advances in interactomics. Low-resolution structural techniques, such as small-angle X-ray scattering (SAXS), can be applied at larger scale, but they miss atomic details. For efficient application to protein-protein complexes, low-resolution information can be combined with theoretical methods that provide energetic description and atomic details of the interactions. Here we present the pyDockSAXS web server (http://life.bsc.es/pid/pydocksaxs) that provides an automatic pipeline for modeling the structure of a protein-protein complex from SAXS data. The method uses FTDOCK to generate rigid-body docking models that are subsequently evaluated by a combination of pyDock energy-based scoring function and their capacity to describe SAXS data. The only required input files are structural models for the interacting partners and a SAXS curve. The server automatically provides a series of structural models for the complex, sorted by the pyDockSAXS scoring function. The user can also upload a previously computed set of docking poses, which opens the possibility to filter the docking solutions by potential interface residues or symmetry restraints. The server is freely available to all users without restriction. PMID:25897115

  2. Retinal Cone Photoreceptors Require Phosducin-Like Protein 1 for G Protein Complex Assembly and Signaling

    PubMed Central

    Tracy, Christopher M.; Kolesnikov, Alexander V.; Blake, Devon R.; Chen, Ching-Kang; Baehr, Wolfgang; Kefalov, Vladimir J.; Willardson, Barry M.

    2015-01-01

    G protein β subunits (Gβ) play essential roles in phototransduction as part of G protein βγ (Gβγ) and regulator of G protein signaling 9 (RGS9)-Gβ5 heterodimers. Both are obligate dimers that rely on the cytosolic chaperone CCT and its co-chaperone PhLP1 to form complexes from their nascent polypeptides. The importance of PhLP1 in the assembly process was recently demonstrated in vivo in a retinal rod-specific deletion of the Phlp1 gene. To test whether this is a general mechanism that also applies to other cell types, we disrupted the Phlp1 gene specifically in mouse cones and measured the effects on G protein expression and cone visual signal transduction. In PhLP1-deficient cones, expression of cone transducin (Gt2) and RGS9-Gβ5 subunits was dramatically reduced, resulting in a 27-fold decrease in sensitivity and a 38-fold delay in cone photoresponse recovery. These results demonstrate the essential role of PhLP1 in cone G protein complex formation. Our findings reveal a common mechanism of Gβγ and RGS9-Gβ5 assembly in rods and cones, highlighting the importance of PhLP1 and CCT-mediated Gβ complex formation in G protein signaling. PMID:25659125

  3. Differences in protein binding and excretion of Triapine and its Fe(III) complex.

    PubMed

    Pelivan, Karla; Miklos, Walter; van Schoonhoven, Sushilla; Koellensperger, Gunda; Gille, Lars; Berger, Walter; Heffeter, Petra; Kowol, Christian R; Keppler, Bernhard K

    2016-07-01

    Triapine has been investigated as anticancer drug in multiple clinical phase I/II trials. Although promising anti-leukemic activity was observed, Triapine was ineffective against solid tumors. The reasons are currently widely unknown. The biological activity of Triapine is strongly connected to its iron complex (Fe-Triapine) which is pharmacologically not investigated. Here, novel analytical tools for Triapine and Fe-Triapine were developed and applied for cell extracts and body fluids of treated mice. Triapine and its iron complex showed a completely different behavior: for Triapine, low protein binding was observed in contrast to fast protein adduct formation of Fe-Triapine. Notably, both drugs were rapidly cleared from the body (serum half-life time <1h). Remarkably, in contrast to Triapine, where (in accordance to clinical data) basically no renal excretion was found, the iron complex was effectively excreted via urine. Moreover, no Fe-Triapine was detected in serum or cytosolic extracts after Triapine treatment. Taken together, our study will help to further understand the biological behavior of Triapine and its Fe-complex and allow the development of novel thiosemicarbazones with pronounced activity against solid tumor types. PMID:26507768

  4. Encounter complexes and dimensionality reduction in protein–protein association

    PubMed Central

    Kozakov, Dima; Li, Keyong; Hall, David R; Beglov, Dmitri; Zheng, Jiefu; Vakili, Pirooz; Schueler-Furman, Ora; Paschalidis, Ioannis Ch; Clore, G Marius; Vajda, Sandor

    2014-01-01

    An outstanding challenge has been to understand the mechanism whereby proteins associate. We report here the results of exhaustively sampling the conformational space in protein–protein association using a physics-based energy function. The agreement between experimental intermolecular paramagnetic relaxation enhancement (PRE) data and the PRE profiles calculated from the docked structures shows that the method captures both specific and non-specific encounter complexes. To explore the energy landscape in the vicinity of the native structure, the nonlinear manifold describing the relative orientation of two solid bodies is projected onto a Euclidean space in which the shape of low energy regions is studied by principal component analysis. Results show that the energy surface is canyon-like, with a smooth funnel within a two dimensional subspace capturing over 75% of the total motion. Thus, proteins tend to associate along preferred pathways, similar to sliding of a protein along DNA in the process of protein-DNA recognition. DOI: http://dx.doi.org/10.7554/eLife.01370.001 PMID:24714491

  5. 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

  6. 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.

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

    PubMed

    Paquet, Françoise; Delalande, Olivier; Goffinont, Stephane; Culard, Françoise; Loth, Karine; Asseline, Ulysse; Castaing, Bertrand; Landon, Celine

    2014-01-01

    In Archaea the two major modes of DNA packaging are wrapping by histone proteins or bending by architectural non-histone proteins. To supplement our knowledge about the binding mode of the different DNA-bending proteins observed across the three domains of life, we present here the first model of a complex in which the monomeric Methanogen Chromosomal protein 1 (MC1) from Euryarchaea binds to the concave side of a strongly bent DNA. In laboratory growth conditions MC1 is the most abundant architectural protein present in Methanosarcina thermophila CHTI55. Like most proteins that strongly bend DNA, MC1 is known to bind in the minor groove. Interaction areas for MC1 and DNA were mapped by Nuclear Magnetic Resonance (NMR) data. The polarity of protein binding was determined using paramagnetic probes attached to the DNA. The first structural model of the DNA-MC1 complex we propose here was obtained by two complementary docking approaches and is in good agreement with the experimental data previously provided by electron microscopy and biochemistry. Residues essential to DNA-binding and -bending were highlighted and confirmed by site-directed mutagenesis. It was found that the Arg25 side-chain was essential to neutralize the negative charge of two phosphates that come very close in response to a dramatic curvature of the DNA. PMID:24558431

  8. Electrostatic Rate Enhancement and Transient Complex of Protein-Protein Association

    PubMed Central

    Alsallaq, Ramzi; Zhou, Huan-Xiang

    2012-01-01

    The association of two proteins is bounded by the rate at which they, via diffusion, find each other while in appropriate relative orientations. Orientational constraints restrict this rate to ~105 – 106 M−1s−1. Proteins with higher association rates generally have complementary electrostatic surfaces; proteins with lower association rates generally are slowed down by conformational changes upon complex formation. Previous studies (Zhou, Biophys. J. 1997;73:2441–2445) have shown that electrostatic enhancement of the diffusion-limited association rate can be accurately modeled by kD = kD0 exp(−*/ kBT), where kD and kD0 are the rates in the presence and absence of electrostatic interactions, respectively, * is the average electrostatic interaction energy in a “transient-complex” ensemble, and kBT is thermal energy. The transient-complex ensemble separates the bound state from the unbound state. Predictions of the transient-complex theory on four protein complexes were found to agree well with experiment when the electrostatic interaction energy was calculated with the linearized Poisson-Boltzmann (PB) equation (Alsallaq and Zhou, Structure 2007, 15:215–224). Here we show that the agreement is further improved when the nonlinear PB equation is used. These predictions are obtained with the dielectric boundary defined as the protein van der Waals surface. When the dielectric boundary is instead specified as the molecular surface, electrostatic interactions in the transient complex become repulsive and are thus predicted to retard association. Together these results demonstrate that the transient-complex theory is predictive of electrostatic rate enhancement and can help parameterize PB calculations. PMID:17932929

  9. 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

  10. 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.

  11. 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. PMID:24016530

  12. 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

  13. 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

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

    PubMed

    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

  15. 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

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

    PubMed

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

    2016-06-01

    NMR structures consist in ensembles of conformers, all satisfying the experimental restraints, which exhibit a certain degree of structural variability. We analyzed here the interface in NMR ensembles of protein-protein heterodimeric complexes and found it to span a wide range of different conservations. The different exhibited conservations do not simply correlate with the size of the systems/interfaces, and are most probably the result of an interplay between different factors, including the quality of experimental data and the intrinsic complex flexibility. In any case, this information is not to be missed when NMR structures of protein-protein complexes are analyzed; especially considering that, as we also show here, the first NMR conformer is usually not the one which best reflects the overall interface. To quantify the interface conservation and to analyze it, we used an approach originally conceived for the analysis and ranking of ensembles of docking models, which has now been extended to directly deal with NMR ensembles. We propose this approach, based on the conservation of the inter-residue contacts at the interface, both for the analysis of the interface in whole ensembles of NMR complexes and for the possible selection of a single conformer as the best representative of the overall interface. In order to make the analyses automatic and fast, we made the protocol available as a web tool at: https://www.molnac.unisa.it/BioTools/consrank/consrank-nmr.html. PMID:26968364

  17. Assignment of protein backbone resonances using connectivity, torsion angles and 13Calpha chemical shifts.

    PubMed

    Morris, Laura C; Valafar, Homayoun; Prestegard, James H

    2004-05-01

    A program is presented which will return the most probable sequence location for a short connected set of residues in a protein given just (13)C(alpha) chemical shifts (delta((13)C(alpha))) and data restricting the phi and psi backbone angles. Data taken from both the BioMagResBank and the Protein Data Bank were used to create a probability density function (PDF) using a multivariate normal distribution in delta((13)C(alpha)), phi, and psi space for each amino acid residue. Extracting and combining probabilities for particular amino acid residues in a short proposed sequence yields a score indicative of the correctness of the proposed assignment. The program is illustrated using several proteins for which structure and (13)C(alpha) chemical shift data are available. PMID:15017135

  18. 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

  19. Identification of short peptide sequences in complex milk protein hydrolysates.

    PubMed

    O'Keeffe, Martina B; FitzGerald, Richard J

    2015-10-01

    Numerous low molecular mass bioactive peptides (BAPs) can be generated during the hydrolysis of bovine milk proteins. Low molecular mass BAP sequences are less likely to be broken down by digestive enzymes and are thus more likely to be active in vivo. However, the identification of short peptides remains a challenge during mass spectrometry (MS) analysis due to issues with the transfer and over-fragmentation of low molecular mass ions. A method is described herein using time-of-flight ESI-MS/MS to effectively fragment and identify short peptides. This includes (a) short synthetic peptides, (b) short peptides within a defined hydrolysate sample, i.e. a prolyl endoproteinase hydrolysate of β-casein and (c) short peptides within a complex hydrolysate, i.e. a Corolase PP digest of sodium caseinate. The methodology may find widespread utilisation in the efficient identification of low molecular mass peptide sequences in food protein hydrolysates. PMID:25872436

  20. 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

  1. 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

  2. Purification and characterization of HIV–human protein complexes

    PubMed Central

    Jäger, Stefanie; Gulbahce, Natali; Cimermancic, Peter; Kane, Joshua; He, Nanhai; Chou, Seemay; D’Orso, Iván; Fernandes, Jason; Jang, Gwendolyn; Frankel, Alan D.; Alber, Tom; Zhou, Qiang; Krogan, Nevan J.

    2011-01-01

    To fully understand how pathogens infect their host and hijack key biological processes, systematic mapping of intra-pathogenic and pathogen–host protein–protein interactions (PPIs) is crucial. Due to the relatively small size of viral genomes (usually around 10–100 proteins), generation of comprehensive host–virus PPI maps using different experimental platforms, including affinity tag purification-mass spectrometry (AP-MS) and yeast two-hybrid (Y2H) approaches, can be achieved. Global maps such as these provide unbiased insight into the molecular mechanisms of viral entry, replication and assembly. However, to date, only two-hybrid methodology has been used in a systematic fashion to characterize viral–host protein–protein interactions, although a deluge of data exists in databases that manually curate from the literature individual host–pathogen PPIs. We will summarize this work and also describe an AP-MS platform that can be used to characterize viral-human protein complexes and discuss its application for the HIV genome. PMID:20708689

  3. The small G-protein MglA connects to the MreB actin cytoskeleton at bacterial focal adhesions

    PubMed Central

    Treuner-Lange, Anke; Macia, Eric; Guzzo, Mathilde; Hot, Edina; Faure, Laura M.; Jakobczak, Beata; Espinosa, Leon; Alcor, Damien; Ducret, Adrien; Keilberg, Daniela; Castaing, Jean Philippe; Lacas Gervais, Sandra; Franco, Michel

    2015-01-01

    In Myxococcus xanthus the gliding motility machinery is assembled at the leading cell pole to form focal adhesions, translocated rearward to propel the cell, and disassembled at the lagging pole. We show that MglA, a Ras-like small G-protein, is an integral part of this machinery. In this function, MglA stimulates the assembly of the motility complex by directly connecting it to the MreB actin cytoskeleton. Because the nucleotide state of MglA is regulated spatially and MglA only binds MreB in the guanosine triphosphate–bound form, the motility complexes are assembled at the leading pole and dispersed at the lagging pole where the guanosine triphosphatase activating protein MglB disrupts the MglA–MreB interaction. Thus, MglA acts as a nucleotide-dependent molecular switch to regulate the motility machinery spatially. The function of MreB in motility is independent of its function in peptidoglycan synthesis, representing a coopted function. Our findings highlight a new function for the MreB cytoskeleton and suggest that G-protein–cytoskeleton interactions are a universally conserved feature. PMID:26169353

  4. Simulating evolution of protein complexes through gene duplication and co-option.

    PubMed

    Haarsma, Loren; Nelesen, Serita; VanAndel, Ethan; Lamine, James; VandeHaar, Peter

    2016-06-21

    We present a model of the evolution of protein complexes with novel functions through gene duplication, mutation, and co-option. Under a wide variety of input parameters, digital organisms evolve complexes of 2-5 bound proteins which have novel functions but whose component proteins are not independently functional. Evolution of complexes with novel functions happens more quickly as gene duplication rates increase, point mutation rates increase, protein complex functional probability increases, protein complex functional strength increases, and protein family size decreases. Evolution of complexity is inhibited when the metabolic costs of making proteins exceeds the fitness gain of having functional proteins, or when point mutation rates get so large the functional proteins undergo deleterious mutations faster than new functional complexes can evolve. PMID:27038665

  5. 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

  6. Mapping energy transfer channels in fucoxanthin-chlorophyll protein complex.

    PubMed

    Gelzinis, Andrius; Butkus, Vytautas; Songaila, Egidijus; Augulis, Ramūnas; Gall, Andrew; Büchel, Claudia; Robert, Bruno; Abramavicius, Darius; Zigmantas, Donatas; Valkunas, Leonas

    2015-02-01

    Fucoxanthin-chlorophyll protein (FCP) is the key molecular complex performing the light-harvesting function in diatoms, which, being a major group of algae, are responsible for up to one quarter of the total primary production on Earth. These photosynthetic organisms contain an unusually large amount of the carotenoid fucoxanthin, which absorbs the light in the blue-green spectral region and transfers the captured excitation energy to the FCP-bound chlorophylls. Due to the large number of fucoxanthins, the excitation energy transfer cascades in these complexes are particularly tangled. In this work we present the two-color two-dimensional electronic spectroscopy experiments on FCP. Analysis of the data using the modified decay associated spectra permits a detailed mapping of the excitation frequency dependent energy transfer flow with a femtosecond time resolution. PMID:25445318

  7. 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.

  8. 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

  9. 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

  10. 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

  11. STATIC AND KINETIC SITE-SPECIFIC PROTEIN-DNA PHOTOCROSSLINKING: ANALYSIS OF BACTERIAL TRANSCRIPTION INITIATION COMPLEXES

    PubMed Central

    Naryshkin, Nikolai; Druzhinin, Sergei; Revyakin, Andrei; Kim, Younggyu; Mekler, Vladimir; Ebright, Richard H.

    2009-01-01

    Static site-specific protein-DNA photocrosslinking permits identification of protein-DNA interactions within multiprotein-DNA complexes. Kinetic site-specific protein-DNA photocrosslinking--involving rapid-quench-flow mixing and pulsed-laser irradiation--permits elucidation of pathways and kinetics of formation of protein-DNA interactions within multiprotein-DNA complexes. We present detailed protocols for application of static and kinetic site-specific protein-DNA photocrosslinking to bacterial transcription initiation complexes. PMID:19378179

  12. Connective-tissue growth factor modulates WNT signalling and interacts with the WNT receptor complex.

    PubMed

    Mercurio, Sara; Latinkic, Branko; Itasaki, Nobue; Krumlauf, Robb; Smith, J C

    2004-05-01

    Connective-tissue growth factor (CTGF) is a member of the CCN family of secreted proteins. CCN family members contain four characteristic domains and exhibit multiple activities: they associate with the extracellular matrix, they can mediate cell adhesion, cell migration and chemotaxis, and they can modulate the activities of peptide growth factors. Many of the effects of CTGF are thought to be mediated by binding to integrins, whereas others may be because of its recently identified ability to interact with BMP4 and TGF beta. We demonstrate, using Xenopus embryos, that CTGF also regulates signalling through the Wnt pathway, in accord with its ability to bind to the Wnt co-receptor LDL receptor-related protein 6 (LRP6). This interaction is likely to occur through the C-terminal (CT) domain of CTGF, which is distinct from the BMP- and TGF beta-interacting domain. Our results define new activities of CTGF and add to the variety of routes through which cells regulate growth factor activity in development, disease and tissue homeostasis. PMID:15105373

  13. 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

  14. Structural Assembly of Multidomain Proteins and Protein Complexes Guided by the Overall Rotational Diffusion Tensor

    PubMed Central

    Ryabov, Yaroslav; Fushman, David

    2008-01-01

    We present a simple and robust approach that uses the overall rotational diffusion tensor as a structural constraint for domain positioning in multidomain proteins and protein-protein complexes. This method offers the possibility to use NMR relaxation data for detailed structure characterization of such systems provided the structures of individual domains are available. The proposed approach extends the concept of using long-range information contained in the overall rotational diffusion tensor. In contrast to the existing approaches, we use both the principal axes and principal values of protein’s rotational diffusion tensor to determine not only the orientation but also the relative positioning of the individual domains in a protein. This is achieved by finding the domain arrangement in a molecule that provides the best possible agreement with all components of the overall rotational diffusion tensor derived from experimental data. The accuracy of the proposed approach is demonstrated for two protein systems with known domain arrangement and parameters of the overall tumbling: the HIV-1 protease homodimer and Maltose Binding Protein. The accuracy of the method and its sensitivity to domain positioning is also tested using computer-generated data for three protein complexes, for which the experimental diffusion tensors are not available. In addition, the proposed method is applied here to determine, for the first time, the structure of both open and closed conformations of Lys48-linked di-ubiquitin chain, where domain motions render impossible accurate structure determination by other methods. The proposed method opens new avenues for improving structure characterization of proteins in solution. PMID:17550252

  15. Localization of kinesin superfamily proteins to the connecting cilium of fish photoreceptors.

    PubMed

    Beech, P L; Pagh-Roehl, K; Noda, Y; Hirokawa, N; Burnside, B; Rosenbaum, J L

    1996-04-01

    Kinesin superfamily proteins (KIFs) are probable motors in vesicular and non-vesicular transport along microtubular tracks. Since a variety of KIFs have been recently identified in the motile flagella of Chlamydomonas, we sought to ascertain whether KIFs are also associated with the connecting cilia of vertebrate rod photoreceptors. As the only structural link between the rod inner segment and the photosensitive rod outer segment, the connecting cilium is thought to be the channel through which all material passes into and out of the outer segment from the rod cell body. We have performed immunological tests on isolated sunfish rod inner-outer segments (RIS-ROS) using two antibodies that recognize the conserved motor domain of numerous KIFs (anti-LAGSE, a peptide antibody, and anti-Klp1 head, generated against the N terminus of Chlamydomonas Klp1) as well as an antibody specific to a neuronal KIF, KIF3A. On immunoblots of RIS-ROS, LAGSE antibody detected a prominent band at approximately 117 kDa, which is likely to be kinesin heavy chain, and Klp1 head antibody detected a single band at approximately 170 kDa; KIF3A antibody detected a polypeptide at approximately 85 kDa which co-migrated with mammalian KIF3A and displayed ATP-dependent release from rod cytoskeletons. Immunofluorescence localizations with anti-LAGSE and anti-Klp1 head antibodies detected epitopes in the axoneme and ellipsoid, and immunoelectron microscopy with the LAGSE antibody showed that the connecting cilium region was particularly antigenic. Immunofluorescence with anti-KIF3A showed prominent labelling of the connecting cilium and the area surrounding its basal body; the outer segment axoneme and parts of the inner segment coincident with microtubules were also labelled. We propose that these putative kinesin superfamily proteins may be involved in the translocation of material between the rod inner and outer segments. PMID:8718680

  16. 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

  17. Partner-Aware Prediction of Interacting Residues in Protein-Protein Complexes from Sequence Data

    PubMed Central

    Ahmad, Shandar; Mizuguchi, Kenji

    2011-01-01

    Computational prediction of residues that participate in protein-protein interactions is a difficult task, and state of the art methods have shown only limited success in this arena. One possible problem with these methods is that they try to predict interacting residues without incorporating information about the partner protein, although it is unclear how much partner information could enhance prediction performance. To address this issue, the two following comparisons are of crucial significance: (a) comparison between the predictability of inter-protein residue pairs, i.e., predicting exactly which residue pairs interact with each other given two protein sequences; this can be achieved by either combining conventional single-protein predictions or making predictions using a new model trained directly on the residue pairs, and the performance of these two approaches may be compared: (b) comparison between the predictability of the interacting residues in a single protein (irrespective of the partner residue or protein) from conventional methods and predictions converted from the pair-wise trained model. Using these two streams of training and validation procedures and employing similar two-stage neural networks, we showed that the models trained on pair-wise contacts outperformed the partner-unaware models in predicting both interacting pairs and interacting single-protein residues. Prediction performance decreased with the size of the conformational change upon complex formation; this trend is similar to docking, even though no structural information was used in our prediction. An example application that predicts two partner-specific interfaces of a protein was shown to be effective, highlighting the potential of the proposed approach. Finally, a preliminary attempt was made to score docking decoy poses using prediction of interacting residue pairs; this analysis produced an encouraging result. PMID:22194998

  18. 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

  19. 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

  20. Antibodies against distinct nuclear matrix proteins are characteristic for mixed connective tissue disease.

    PubMed Central

    Habets, W J; de Rooij, D J; Salden, M H; Verhagen, A P; van Eekelen, C A; van de Putte, L B; van Venrooij, W J

    1983-01-01

    Specific nuclear proteins, separated according to their molecular weight (mol. wt) by polyacrylamide gel electrophoresis (PAGE) and subsequently transferred to nitrocellulose sheets, are able to bind antibodies in sera from patients suffering from different types of connective tissue diseases. Antibodies against a characteristic set of nuclear protein antigens are found in sera from patients with mixed connective tissue disease (MCTD). Screening of 21 MCTD sera revealed a typical immunoblot pattern with major protein antigens of mol. wt 70,000 (20/21) (not identical with the Scl-70 antigen characteristic for scleroderma), mol. wt 31,000 (17/21), two proteins around mol. wt 23,000 (15/21) and two around mol. wt 19,000 (10/21). The 70,000, 23,000 and 19,000 antigens appeared to be rather insoluble nuclear proteins (i.e. components of the nuclear matrix). On behalf of their structural character they were present in nuclei from several types of cells but only in low amounts detectable in salt extracts of thymus acetone powder. The presence of antibodies directed against the mol. wt 70,000 antigen correlated strongly with the diagnosis of MCTD. This 70,000 antigen is not identical with the RNP antigen, a soluble ribonuclease sensitive ribonucleoprotein, since antibodies against nuclear RNP can be separated from anti-nuclear matrix antibodies by affinity chromatography using immobilized thymus salt extract. The distinct character of soluble nuclear RNP and structural nuclear matrix antigens is further supported by the fact that from 14 other anti-RNP sera obtained from patients with systemic lupus erythematosus (SLE), only three contained antibodies against the mol. wt 70,000 protein. Since the immunoblot pattern obtained with MCTD sera mostly was clearly distinguishable from the patterns obtained with sera from patients with related connective tissue diseases our results suggest that the immunoblotting technique might be useful as a diagnostic tool and support the

  1. A novel algorithm for detecting protein complexes with the breadth first search.

    PubMed

    Tang, Xiwei; Wang, Jianxin; Li, Min; He, Yiming; Pan, Yi

    2014-01-01

    Most biological processes are carried out by protein complexes. A substantial number of false positives of the protein-protein interaction (PPI) data can compromise the utility of the datasets for complexes reconstruction. In order to reduce the impact of such discrepancies, a number of data integration and affinity scoring schemes have been devised. The methods encode the reliabilities (confidence) of physical interactions between pairs of proteins. The challenge now is to identify novel and meaningful protein complexes from the weighted PPI network. To address this problem, a novel protein complex mining algorithm ClusterBFS (Cluster with Breadth-First Search) is proposed. Based on the weighted density, ClusterBFS detects protein complexes of the weighted network by the breadth first search algorithm, which originates from a given seed protein used as starting-point. The experimental results show that ClusterBFS performs significantly better than the other computational approaches in terms of the identification of protein complexes. PMID:24818139

  2. Nanobody-targeted E3-ubiquitin ligase complex degrades nuclear proteins

    PubMed Central

    Ju Shin, Yeong; Kyun Park, Seung; Jung Jung, Yoo; Na Kim, Ye; Sung Kim, Ki; Kyu Park, Ok; Kwon, Seung-Hae; Ho Jeon, Sung; Trinh, Le A.; Fraser, Scott E.; Kee, Yun; Joon Hwang, Byung

    2015-01-01

    Targeted protein degradation is a powerful tool in determining the function of specific proteins or protein complexes. We fused nanobodies to SPOP, an adaptor protein of the Cullin-RING E3 ubiquitin ligase complex, resulting in rapid ubiquitination and subsequent proteasome-dependent degradation of specific nuclear proteins in mammalian cells and zebrafish embryos. This approach is easily modifiable, as substrate specificity is conferred by an antibody domain that can be adapted to target virtually any protein. PMID:26373678

  3. Polysaccharide-Protein Complexes in a Coarse-Grained Model.

    PubMed

    Poma, Adolfo B; Chwastyk, Mateusz; Cieplak, Marek

    2015-09-10

    We construct two variants of coarse-grained models of three hexaoses: one based on the centers of mass of the monomers and the other associated with the C4 atoms. The latter is found to be better defined and more suitable for studying interactions with proteins described within α-C based models. We determine the corresponding effective stiffness constants through all-atom simulations and two statistical methods. One method is the Boltzmann inversion (BI) and the other, named energy-based (EB), involves direct monitoring of energies as a function of the variables that define the stiffness potentials. The two methods are generally consistent in their account of the stiffness. We find that the elastic constants differ between the hexaoses and are noticeably different from those determined for the crystalline cellulose Iβ. The nonbonded couplings through hydrogen bonds between different sugar molecules are modeled by the Lennard-Jones potentials and are found to be stronger than the hydrogen bonds in proteins. We observe that the EB method agrees with other theoretical and experimental determinations of the nonbonded parameters much better than BI. We then consider the hexaose-Man5B catalytic complexes and determine the contact energies between their the C4-α-C atoms. These interactions are found to be stronger than the proteinic hydrogen bonds: about four times as strong for cellohexaose and two times for mannohexaose. The fluctuational dynamics of the coarse-grained complexes are found to be compatible with previous all-atom studies by Bernardi et al. PMID:26291477

  4. 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

  5. Nucleocytoplasmic Transport of RNAs and RNA-Protein Complexes.

    PubMed

    Sloan, Katherine E; Gleizes, Pierre-Emmanuel; Bohnsack, Markus T

    2016-05-22

    RNAs and ribonucleoprotein complexes (RNPs) play key roles in mediating and regulating gene expression. In eukaryotes, most RNAs are transcribed, processed and assembled with proteins in the nucleus and then either function in the cytoplasm or also undergo a cytoplasmic phase in their biogenesis. This compartmentalization ensures that sequential steps in gene expression and RNP production are performed in the correct order and it allows important quality control mechanisms that prevent the involvement of aberrant RNAs/RNPs in these cellular pathways. The selective exchange of RNAs/RNPs between the nucleus and cytoplasm is enabled by nuclear pore complexes, which function as gateways between these compartments. RNA/RNP transport is facilitated by a range of nuclear transport receptors and adaptors, which are specifically recruited to their cargos and mediate interactions with nucleoporins to allow directional translocation through nuclear pore complexes. While some transport factors are only responsible for the export/import of a certain class of RNA/RNP, others are multifunctional and, in the case of large RNPs, several export factors appear to work together to bring about export. Recent structural studies have revealed aspects of the mechanisms employed by transport receptors to enable specific cargo recognition, and genome-wide approaches have provided the first insights into the diverse composition of pre-mRNPs during export. Furthermore, the regulation of RNA/RNP export is emerging as an important means to modulate gene expression under stress conditions and in disease. PMID:26434509

  6. 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

  7. Detection of an endothelin-1-binding protein complex by low temperature SDS-PAGE

    SciTech Connect

    Takasuka, T.; Horii, I.; Furuichi, Y.; Watanabe, T. )

    1991-04-15

    We found that the complex of ET-1 and its binding protein was stable enough to be separated by SDS-PAGE when electrophoresis was run at a low temperature. Cross-linking was not necessary for the detection of {sup 125}I-ET-1 and its binding protein complex by autoradiography. This simple method could be used in qualitative (estimation of apparent molecular weight of ET-1 binding protein) and quantitative (determination of relative content of ET-binding protein) analysis of the ET-binding protein complex. ET-binding protein complexes of various animal species and organs were investigated by this method.

  8. 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

  9. Network Reconstruction Based on Proteomic Data and Prior Knowledge of Protein Connectivity Using Graph Theory

    PubMed Central

    Stavrakas, Vassilis; 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

  10. Excitation energy transfer in photosynthetic protein-pigment complexes

    NASA Astrophysics Data System (ADS)

    Yeh, Shu-Hao

    Quantum biology is a relatively new research area which investigates the rules that quantum mechanics plays in biology. One of the most intriguing systems in this field is the coherent excitation energy transport (EET) in photosynthesis. In this document I will discuss the theories that are suitable for describing the photosynthetic EET process and the corresponding numerical results on several photosynthetic protein-pigment complexes (PPCs). In some photosynthetic EET processes, because of the electronic coupling between the chromophores within the system is about the same order of magnitude as system-bath coupling (electron-phonon coupling), a non-perturbative method called hierarchy equation of motion (HEOM) is applied to study the EET dynamics. The first part of this thesis includes brief introduction and derivation to the HEOM approach. The second part of this thesis the HEOM method will be applied to investigate the EET process within the B850 ring of the light harvesting complex 2 (LH2) from purple bacteria, Rhodopseudomonas acidophila. The dynamics of the exciton population and coherence will be analyzed under different initial excitation configurations and temperatures. Finally, how HEOM can be implemented to simulate the two-dimensional electronic spectra of photosynthetic PPCs will be discussed. Two-dimensional electronic spectroscopy is a crucial experimental technique to probe EET dynamics in multi-chromophoric systems. The system we are interested in is the 7-chromophore Fenna-Matthews-Olson (FMO) complex from green sulfur bacteria, Prosthecochloris aestuarii. Recent crystallographic studies report the existence of an additional (eighth) chromophore in some of the FMO monomers. By applying HEOM we are able to calculate the two-dimensional electronic spectra of the 7-site and 8-site FMO complexes and investigate the functionality of the eighth chromophore.

  11. 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

  12. Hox Proteins Coordinate Motor Neuron Differentiation and Connectivity Programs through Ret/Gfrα Genes.

    PubMed

    Catela, Catarina; Shin, Maggie M; Lee, David H; Liu, Jeh-Ping; Dasen, Jeremy S

    2016-03-01

    The accuracy of neural circuit assembly relies on the precise spatial and temporal control of synaptic specificity determinants during development. Hox transcription factors govern key aspects of motor neuron (MN) differentiation; however, the terminal effectors of their actions are largely unknown. We show that Hox/Hox cofactor interactions coordinate MN subtype diversification and connectivity through Ret/Gfrα receptor genes. Hox and Meis proteins determine the levels of Ret in MNs and define the intrasegmental profiles of Gfrα1 and Gfrα3 expression. Loss of Ret or Gfrα3 leads to MN specification and innervation defects similar to those observed in Hox mutants, while expression of Ret and Gfrα1 can bypass the requirement for Hox genes during MN pool differentiation. These studies indicate that Hox proteins contribute to neuronal fate and muscle connectivity through controlling the levels and pattern of cell surface receptor expression, consequently gating the ability of MNs to respond to limb-derived instructive cues. PMID:26904955

  13. Comparison of tertiary structures of proteins in protein-protein complexes with unbound forms suggests prevalence of allostery in signalling proteins

    PubMed Central

    2012-01-01

    Background Most signalling and regulatory proteins participate in transient protein-protein interactions during biological processes. They usually serve as key regulators of various cellular processes and are often stable in both protein-bound and unbound forms. Availability of high-resolution structures of their unbound and bound forms provides an opportunity to understand the molecular mechanisms involved. In this work, we have addressed the question “What is the nature, extent, location and functional significance of structural changes which are associated with formation of protein-protein complexes?” Results A database of 76 non-redundant sets of high resolution 3-D structures of protein-protein complexes, representing diverse functions, and corresponding unbound forms, has been used in this analysis. Structural changes associated with protein-protein complexation have been investigated using structural measures and Protein Blocks description. Our study highlights that significant structural rearrangement occurs on binding at the interface as well as at regions away from the interface to form a highly specific, stable and functional complex. Notably, predominantly unaltered interfaces interact mainly with interfaces undergoing substantial structural alterations, revealing the presence of at least one structural regulatory component in every complex. Interestingly, about one-half of the number of complexes, comprising largely of signalling proteins, show substantial localized structural change at surfaces away from the interface. Normal mode analysis and available information on functions on some of these complexes suggests that many of these changes are allosteric. This change is largely manifest in the proteins whose interfaces are altered upon binding, implicating structural change as the possible trigger of allosteric effect. Although large-scale studies of allostery induced by small-molecule effectors are available in literature, this is, to our

  14. 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.

  15. 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.

  16. 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

  17. 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

  18. Building Disease-Specific Drug-Protein Connectivity Maps from Molecular Interaction Networks and PubMed Abstracts

    PubMed Central

    Li, Jiao; Zhu, Xiaoyan; Chen, Jake Yue

    2009-01-01

    The recently proposed concept of molecular connectivity maps enables researchers to integrate experimental measurements of genes, proteins, metabolites, and drug compounds under similar biological conditions. The study of these maps provides opportunities for future toxicogenomics and drug discovery applications. We developed a computational framework to build disease-specific drug-protein connectivity maps. We integrated gene/protein and drug connectivity information based on protein interaction networks and literature mining, without requiring gene expression profile information derived from drug perturbation experiments on disease samples. We described the development and application of this computational framework using Alzheimer's Disease (AD) as a primary example in three steps. First, molecular interaction networks were incorporated to reduce bias and improve relevance of AD seed proteins. Second, PubMed abstracts were used to retrieve enriched drug terms that are indirectly associated with AD through molecular mechanistic studies. Third and lastly, a comprehensive AD connectivity map was created by relating enriched drugs and related proteins in literature. We showed that this molecular connectivity map development approach outperformed both curated drug target databases and conventional information retrieval systems. Our initial explorations of the AD connectivity map yielded a new hypothesis that diltiazem and quinidine may be investigated as candidate drugs for AD treatment. Molecular connectivity maps derived computationally can help study molecular signature differences between different classes of drugs in specific disease contexts. To achieve overall good data coverage and quality, a series of statistical methods have been developed to overcome high levels of data noise in biological networks and literature mining results. Further development of computational molecular connectivity maps to cover major disease areas will likely set up a new model for

  19. Building disease-specific drug-protein connectivity maps from molecular interaction networks and PubMed abstracts.

    PubMed

    Li, Jiao; Zhu, Xiaoyan; Chen, Jake Yue

    2009-07-01

    The recently proposed concept of molecular connectivity maps enables researchers to integrate experimental measurements of genes, proteins, metabolites, and drug compounds under similar biological conditions. The study of these maps provides opportunities for future toxicogenomics and drug discovery applications. We developed a computational framework to build disease-specific drug-protein connectivity maps. We integrated gene/protein and drug connectivity information based on protein interaction networks and literature mining, without requiring gene expression profile information derived from drug perturbation experiments on disease samples. We described the development and application of this computational framework using Alzheimer's Disease (AD) as a primary example in three steps. First, molecular interaction networks were incorporated to reduce bias and improve relevance of AD seed proteins. Second, PubMed abstracts were used to retrieve enriched drug terms that are indirectly associated with AD through molecular mechanistic studies. Third and lastly, a comprehensive AD connectivity map was created by relating enriched drugs and related proteins in literature. We showed that this molecular connectivity map development approach outperformed both curated drug target databases and conventional information retrieval systems. Our initial explorations of the AD connectivity map yielded a new hypothesis that diltiazem and quinidine may be investigated as candidate drugs for AD treatment. Molecular connectivity maps derived computationally can help study molecular signature differences between different classes of drugs in specific disease contexts. To achieve overall good data coverage and quality, a series of statistical methods have been developed to overcome high levels of data noise in biological networks and literature mining results. Further development of computational molecular connectivity maps to cover major disease areas will likely set up a new model for

  20. 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

  1. 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

  2. Protein biotinylation visualized by a complex structure of biotin protein ligase with a substrate.

    PubMed

    Bagautdinov, Bagautdin; Matsuura, Yoshinori; Bagautdinova, Svetlana; Kunishima, Naoki

    2008-05-23

    Biotin protein ligase (BPL) catalyzes the biotinylation of the biotin carboxyl carrier protein (BCCP) only at a special lysine residue. Here we report the first structure of BPL.BCCP complex crystals, which are prepared using two BPL mutants: R48A and R48A/K111A. From a detailed structural characterization, it is likely that the mutants retain functionality as enzymes but have a reduced activity to produce the reaction intermediate biotinyl-5'-AMP. The observed biotin and partly disordered ATP in the mutant structures may act as a non-reactive analog of the substrates or biotinyl-5'-AMP, thereby providing the complex crystals. The four crystallographically independent BPL.BCCP complexes obtained can be classified structurally into three groups: the formation stages 1 and 2 with apo-BCCP and the product stage with biotinylated holo-BCCP. Residues responsible for the complex formation as well as for the biotinylation reaction have been identified. The C-terminal domain of BPL shows especially large conformational changes to accommodate BCCP, suggesting its functional importance. The formation stage 1 complex shows the closest distance between the carboxyl carbon of biotin and the special lysine of BCCP, suggesting its relevance to the unobserved reaction stage. Interestingly, bound ATP and biotin are also seen in the product stage, indicating that the substrates may be recruited into the product stage complex before the release of holo-BCCP, probably for the next reaction cycle. The existence of formation and product stages before and after the reaction stage would be favorable to ensure both the reaction efficiency and the extreme substrate specificity of the biotinylation reaction. PMID:18372281

  3. 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

  4. Mitogen activated protein kinase at the nuclear pore complex

    PubMed Central

    Faustino, Randolph S; Maddaford, Thane G; Pierce, Grant N

    2011-01-01

    Abstract Mitogen activated protein (MAP) kinases control eukaryotic proliferation, and import of kinases into the nucleus through the nuclear pore complex (NPC) can influence gene expression to affect cellular growth, cell viability and homeostatic function. The NPC is a critical regulatory checkpoint for nucleocytoplasmic traffic that regulates gene expression and cell growth, and MAP kinases may be physically associated with the NPC to modulate transport. In the present study, highly enriched NPC fractions were isolated and investigated for associated kinases and/or activity. Endogenous kinase activity was identified within the NPC fraction, which phosphorylated a 30 kD nuclear pore protein. Phosphomodification of this nucleoporin, here termed Nup30, was inhibited by apigenin and PD-98059, two MAP kinase antagonists as well as with SB-202190, a pharmacological blocker of p38. Furthermore, high throughput profiling of enriched NPCs revealed constitutive presence of all members of the MAP kinase family, extracellular regulated kinases (ERK), p38 and Jun N-terminal kinase. The NPC thus contains a spectrum of associated MAP kinases that suggests an intimate role for ERK and p38 in regulation of nuclear pore function. PMID:20497490

  5. Arabidopsis flower development-of protein complexes, targets, and transport.

    PubMed

    Becker, Annette; Ehlers, Katrin

    2016-03-01

    Tremendous progress has been achieved over the past 25 years or more of research on the molecular mechanisms of floral organ identity, patterning, and development. While collections of floral homeotic mutants of Antirrhinum majus laid the foundation already at the beginning of the previous century, it was the genetic analysis of these mutants in A. majus and Arabidopsis thaliana that led to the development of the ABC model of floral organ identity more than 20 years ago. This intuitive model kick-started research focused on the genetic mechanisms regulating flower development, using mainly A. thaliana as a model plant. In recent years, interactions among floral homeotic proteins have been elucidated, and their direct and indirect target genes are known to a large extent. Here, we provide an overview over the advances in understanding the molecular mechanism orchestrating A. thaliana flower development. We focus on floral homeotic protein complexes, their target genes, evidence for their transport in floral primordia, and how these new results advance our view on the processes downstream of floral organ identity, such as organ boundary formation or floral organ patterning. PMID:25845756

  6. 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

  7. 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

  8. 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. PMID:26298638

  9. 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.

  10. 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.

  11. 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

  12. Disparity in Frontal Lobe Connectivity on a Complex Bimanual Motor Task Aids in Classification of Operator Skill Level.

    PubMed

    Andreu-Perez, Javier; Leff, Daniel Richard; Shetty, Kunal; Darzi, Ara; Yang, Guang-Zhong

    2016-06-01

    Objective metrics of technical performance (e.g., dexterity, time, and path length) are insufficient to fully characterize operator skill level, which may be encoded deep within neural function. Unlike reports that capture plasticity across days or weeks, this articles studies long-term plasticity in functional connectivity that occurs over years of professional task practice. Optical neuroimaging data are acquired from professional surgeons of varying experience on a complex bimanual coordination task with the aim of investigating learning-related disparity in frontal lobe functional connectivity that arises as a consequence of motor skill level. The results suggest that prefrontal and premotor seed connectivity is more critical during naïve versus expert performance. Given learning-related differences in connectivity, a least-squares support vector machine with a radial basis function kernel is employed to evaluate skill level using connectivity data. The results demonstrate discrimination of operator skill level with accuracy ≥0.82 and Multiclass Matthew's Correlation Coefficient ≥0.70. Furthermore, these indices are improved when local (i.e., within-region) rather than inter-regional (i.e., between-region) frontal connectivity is considered (p = 0.002). The results suggest that it is possible to classify operator skill level with good accuracy from functional connectivity data, upon which objective assessment and neurofeedback may be used to improve operator performance during technical skill training. PMID:26899241

  13. 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

  14. 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

  15. Tandem Affinity Purification Combined with Mass Spectrometry to Identify Components of Protein Complexes

    PubMed Central

    Kaiser, Peter; Meierhofer, David; Wang, Xiaorong; Huang, Lan

    2011-01-01

    Most biological processes are governed by multiprotein complexes rather than individual proteins. Identification of protein complexes therefore is becoming increasingly important to gain a molecular understanding of cells and organisms. Mass spectrometry–based proteomics combined with affinity-tag-based protein purification is one of the most effective strategies to isolate and identify protein complexes. The development of tandem-affinity purification approaches has revolutionized proteomics experiments. These two-step affinity purification strategies allow rapid, effective purification of protein complexes and, at the same time, minimize background. Identification of even very low-abundant protein complexes with modern sensitive mass spectrometers has become routine. Here, we describe two general strategies for tandem-affinity purification followed by mass spectrometric identification of protein complexes. PMID:18370112

  16. Functional mammalian spliceosomal complex E contains SMN complex proteins in addition to U1 and U2 snRNPs

    PubMed Central

    Makarov, Evgeny M.; Owen, Nicholas; Bottrill, Andrew; Makarova, Olga V.

    2012-01-01

    Spliceosomes remove introns from primary gene transcripts. They assemble de novo on each intron through a series of steps that involve the incorporation of five snRNP particles and multiple non-snRNP proteins. In mammals, all the intermediate complexes have been characterized on one transcript (MINX), with the exception of the very first, complex E. We have purified this complex by two independent procedures using antibodies to either U1-A or PRPF40A proteins, which are known to associate at an early stage of assembly. We demonstrate that the purified complexes are functional in splicing using commitment assays. These complexes contain components expected to be in the E complex and a number of previously unrecognized factors, including survival of motor neurons (SMN) and proteins of the SMN-associated complex. Depletion of the SMN complex proteins from nuclear extracts inhibits formation of the E complex and causes non-productive complexes to accumulate. This suggests that the SMN complex stabilizes the association of U1 and U2 snRNPs with pre-mRNA. In addition, the antibody to PRPF40A precipitated U2 snRNPs from nuclear extracts, indicating that PRPF40A associates with U2 snRNPs. PMID:22110043

  17. New Markov-Shannon Entropy models to assess connectivity quality in complex networks: from molecular to cellular pathway, Parasite-Host, Neural, Industry, and Legal-Social networks.

    PubMed

    Riera-Fernández, Pablo; Munteanu, Cristian R; Escobar, Manuel; Prado-Prado, Francisco; Martín-Romalde, Raquel; Pereira, David; Villalba, Karen; Duardo-Sánchez, Aliuska; González-Díaz, Humberto

    2012-01-21

    : Metabolic networks (72.3%), Parasite-Host networks (93.3%), CoCoMac brain cortex co-activation network (89.6%), NW Spain fasciolosis spreading network (97.2%), Spanish financial law network (89.9%) and World trade network for Intelligent & Active Food Packaging (92.8%). In order to seek these models, we studied an average of 55,388 pairs of nodes in each model and a total of 332,326 pairs of nodes in all models. Finally, this method was used to solve a more complicated problem. A model was developed to score the connectivity quality in the Drug-Target network of US FDA approved drugs. In this last model the θ(k) values were calculated for three types of molecular networks representing different levels of organization: drug molecular graphs (atom-atom bonds), protein residue networks (amino acid interactions), and drug-target network (compound-protein binding). The overall accuracy of this model was 76.3%. This work opens a new door to the computational reevaluation of network connectivity quality (collation) for complex systems in molecular, biomedical, technological, and legal-social sciences as well as in world trade and industry. PMID:22037044

  18. 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

  19. 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

  20. 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

  1. Elongation factor-1A1 is a novel substrate of the protein phosphatase 1-TIMAP complex.

    PubMed

    Boratkó, Anita; Péter, Margit; Thalwieser, Zsófia; Kovács, Előd; Csortos, Csilla

    2015-12-01

    TIMAP (TGF-β inhibited membrane associated protein) is a protein phosphatase 1 (PP1) regulatory subunit highly abundant in endothelial cells and it is involved in the maintenance of pulmonary endothelial barrier function. It localizes mainly in the plasma membrane, but it is also present in the nuclei and cytoplasm. Direct interaction of TIMAP with the eukaryotic elongation factor 1 A1 (eEF1A1) is shown by pull-down, LC-MS/MS, Far-Western and immunoprecipitations. In connection with the so called moonlighting functions of the elongation factor, eEF1A is thought to establish protein-protein interactions through a transcription-dependent nuclear export motif, TD-NEM, and to aid nuclear export of TD-NEM containing proteins. We found that a TD-NEM-like motif of TIMAP has a critical role in its specific binding to eEF1A1. However, eEF1A1 is not or not exclusively responsible for the nuclear export of TIMAP. On the contrary, TIMAP seems to regulate membrane localization of eEF1A1 as the elongation factor co-localized with TIMAP in the plasma membrane fraction of control endothelial cells, but it has disappeared from the membrane in TIMAP depleted cells. It is demonstrated that membrane localization of eEF1A1 depends on the phosphorylation state of its Thr residue(s); and ROCK phosphorylated eEF1A1 is a novel substrate for TIMAP-PP1 underlining the complex regulatory role of TIMAP in the endothelium. The elongation factor seems to be involved in the regulation of endothelial cell attachment and spreading as silencing of eEF1A1 positively affected these processes which were monitored by transendothelial resistance measurements. PMID:26497934

  2. Methods for protein complex prediction and their contributions towards understanding the organisation, function and dynamics of complexes.

    PubMed

    Srihari, Sriganesh; Yong, Chern Han; Patil, Ashwini; Wong, Limsoon

    2015-09-14

    Complexes of physically interacting proteins constitute fundamental functional units responsible for driving biological processes within cells. A faithful reconstruction of the entire set of complexes is therefore essential to understand the functional organisation of cells. In this review, we discuss the key contributions of computational methods developed till date (approximately between 2003 and 2015) for identifying complexes from the network of interacting proteins (PPI network). We evaluate in depth the performance of these methods on PPI datasets from yeast, and highlight their limitations and challenges, in particular at detecting sparse and small or sub-complexes and discerning overlapping complexes. We describe methods for integrating diverse information including expression profiles and 3D structures of proteins with PPI networks to understand the dynamics of complex formation, for instance, of time-based assembly of complex subunits and formation of fuzzy complexes from intrinsically disordered proteins. Finally, we discuss methods for identifying dysfunctional complexes in human diseases, an application that is proving invaluable to understand disease mechanisms and to discover novel therapeutic targets. We hope this review aptly commemorates a decade of research on computational prediction of complexes and constitutes a valuable reference for further advancements in this exciting area. PMID:25913176

  3. 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

  4. KOSMOS: a universal morph server for nucleic acids, proteins and their complexes

    PubMed Central

    Seo, Sangjae; Kim, Moon Ki

    2012-01-01

    KOSMOS is the first online morph server to be able to address the structural dynamics of DNA/RNA, proteins and even their complexes, such as ribosomes. The key functions of KOSMOS are the harmonic and anharmonic analyses of macromolecules. In the harmonic analysis, normal mode analysis (NMA) based on an elastic network model (ENM) is performed, yielding vibrational modes and B-factor calculations, which provide insight into the potential biological functions of macromolecules based on their structural features. Anharmonic analysis involving elastic network interpolation (ENI) is used to generate plausible transition pathways between two given conformations by optimizing a topology-oriented cost function that guarantees a smooth transition without steric clashes. The quality of the computed pathways is evaluated based on their various facets, including topology, energy cost and compatibility with the NMA results. There are also two unique features of KOSMOS that distinguish it from other morph servers: (i) the versatility in the coarse-graining methods and (ii) the various connection rules in the ENM. The models enable us to analyze macromolecular dynamics with the maximum degrees of freedom by combining a variety of ENMs from full-atom to coarse-grained, backbone and hybrid models with one connection rule, such as distance-cutoff, number-cutoff or chemical-cutoff. KOSMOS is available at http://bioengineering.skku.ac.kr/kosmos. PMID:22669912

  5. KOSMOS: a universal morph server for nucleic acids, proteins and their complexes.

    PubMed

    Seo, Sangjae; Kim, Moon Ki

    2012-07-01

    KOSMOS is the first online morph server to be able to address the structural dynamics of DNA/RNA, proteins and even their complexes, such as ribosomes. The key functions of KOSMOS are the harmonic and anharmonic analyses of macromolecules. In the harmonic analysis, normal mode analysis (NMA) based on an elastic network model (ENM) is performed, yielding vibrational modes and B-factor calculations, which provide insight into the potential biological functions of macromolecules based on their structural features. Anharmonic analysis involving elastic network interpolation (ENI) is used to generate plausible transition pathways between two given conformations by optimizing a topology-oriented cost function that guarantees a smooth transition without steric clashes. The quality of the computed pathways is evaluated based on their various facets, including topology, energy cost and compatibility with the NMA results. There are also two unique features of KOSMOS that distinguish it from other morph servers: (i) the versatility in the coarse-graining methods and (ii) the various connection rules in the ENM. The models enable us to analyze macromolecular dynamics with the maximum degrees of freedom by combining a variety of ENMs from full-atom to coarse-grained, backbone and hybrid models with one connection rule, such as distance-cutoff, number-cutoff or chemical-cutoff. KOSMOS is available at http://bioengineering.skku.ac.kr/kosmos. PMID:22669912

  6. 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

  7. 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…

  8. Identifying protein complexes from interaction networks based on clique percolation and distance restriction

    PubMed Central

    2010-01-01

    Background Identification of protein complexes in large interaction networks is crucial to understand principles of cellular organization and predict protein functions, which is one of the most important issues in the post-genomic era. Each protein might be subordinate multiple protein complexes in the real protein-protein interaction networks. Identifying overlapping protein complexes from protein-protein interaction networks is a considerable research topic. Result As an effective algorithm in identifying overlapping module structures, clique percolation method (CPM) has a wide range of application in social networks and biological networks. However, the recognition accuracy of algorithm CPM is lowly. Furthermore, algorithm CPM is unfit to identifying protein complexes with meso-scale when it applied in protein-protein interaction networks. In this paper, we propose a new topological model by extending the definition of k-clique community of algorithm CPM and introduced distance restriction, and develop a novel algorithm called CP-DR based on the new topological model for identifying protein complexes. In this new algorithm, the protein complex size is restricted by distance constraint to conquer the shortcomings of algorithm CPM. The algorithm CP-DR is applied to the protein interaction network of Sacchromyces cerevisiae and identifies many well known complexes. Conclusion The proposed algorithm CP-DR based on clique percolation and distance restriction makes it possible to identify dense subgraphs in protein interaction networks, a large number of which correspond to known protein complexes. Compared to algorithm CPM, algorithm CP-DR has more outstanding performance. PMID:21047377

  9. The Network Organization of Cancer-associated Protein Complexes in Human Tissues

    NASA Astrophysics Data System (ADS)

    Zhao, Jing; Lee, Sang Hoon; Huss, Mikael; Holme, Petter

    2013-04-01

    Differential gene expression profiles for detecting disease genes have been studied intensively in systems biology. However, it is known that various biological functions achieved by proteins follow from the ability of the protein to form complexes by physically binding to each other. In other words, the functional units are often protein complexes rather than individual proteins. Thus, we seek to replace the perspective of disease-related genes by disease-related complexes, exemplifying with data on 39 human solid tissue cancers and their original normal tissues. To obtain the differential abundance levels of protein complexes, we apply an optimization algorithm to genome-wide differential expression data. From the differential abundance of complexes, we extract tissue- and cancer-selective complexes, and investigate their relevance to cancer. The method is supported by a clustering tendency of bipartite cancer-complex relationships, as well as a more concrete and realistic approach to disease-related proteomics.

  10. 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...

  11. Stability and Immunogenicity of Hypoallergenic Peanut Protein-Polyphenol Complexes during In Vitro Pepsin Diges

    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...

  12. 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

  13. Assembly of nuclear pore complexes mediated by major vault protein.

    PubMed

    Vollmar, Friederike; Hacker, Christian; Zahedi, René-Peiman; Sickmann, Albert; Ewald, Andrea; Scheer, Ulrich; Dabauvalle, Marie-Christine

    2009-03-15

    During interphase growth of eukaryotic cells, nuclear pore complexes (NPCs) are continuously incorporated into the intact nuclear envelope (NE) by mechanisms that are largely unknown. De novo formation of NPCs involves local fusion events between the inner and outer nuclear membrane, formation of a transcisternal membranous channel of defined diameter and the coordinated assembly of hundreds of nucleoporins into the characteristic NPC structure. Here we have used a cell-free system based on Xenopus egg extract, which allows the experimental separation of nuclear-membrane assembly and NPC formation. Nuclei surrounded by a closed double nuclear membrane, but devoid of NPCs, were first reconstituted from chromatin and a specific membrane fraction. Insertion of NPCs into the preformed pore-free nuclei required cytosol containing soluble nucleoporins or nucleoporin subcomplexes and, quite unexpectedly, major vault protein (MVP). MVP is the main component of vaults, which are ubiquitous barrel-shaped particles of enigmatic function. Our results implicate MVP, and thus also vaults, in NPC biogenesis and provide a functional explanation for the association of a fraction of vaults with the NE and specifically with NPCs in intact cells. PMID:19240118

  14. 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.

  15. 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

  16. 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. PMID:26492574

  17. 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

  18. 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

  19. SARConnect: A Tool to Interrogate the Connectivity Between Proteins, Chemical Structures and Activity Data

    PubMed Central

    Eriksson, Mats; Nilsson, Ingemar; Kogej, Thierry; Southan, Christopher; Johansson, Martin; Tyrchan, Christian; Muresan, Sorel; Blomberg, Niklas; Bjäreland, Marcus

    2012-01-01

    Abstract The access and use of large-scale structure-activity relationships (SAR) is increasing as the range of targets and availability of bioactive compound-to-protein mappings expands. However, effective exploitation requires merging and normalisation of activity data, mappings to target classifications as well as visual display of chemical structure relationships. This work describes the development of the application “SARConnect” to address these issues. We discuss options for delivery and analysis of large-scale SAR data together with a set of use-cases to illustrate the design choices and utility. The main activity sources of ChEMBL,1 GOSTAR2 and AstraZeneca’s internal system IBIS, had already been integrated in Chemistry Connect.3 For target relationships we selected human UniProtKB/Swiss-Prot4 as our primary source of a heuristic target classification. Similarly, to explore chemical relationships we combined several methods for framework and scaffold analysis into a unified, hierarchical classification where ease of navigation was the primary goal. An application was built on TIBCO Spotfire to retrieve data for visual display. Consequently, users can explore relationships between target, activity and structure across internal, external and commercial sources that encompass approximately 3 million compounds, 2000 human proteins and 10 million activity values. Examples showing the utility of the application are given. PMID:23308082

  20. Mining connections between chemicals, proteins, and diseases extracted from Medline annotations

    PubMed Central

    Baker, Nancy C.; Hemminger, Bradley M.

    2010-01-01

    The biomedical literature is an important source of information about the biological activity and effects of chemicals. We present an application that extracts terms indicating biological activity of chemicals from Medline records, associates them with chemical name and stores the terms in a repository called ChemoText. We describe the construction of ChemoText and then demonstrate its utility in drug research by employing Swanson’s ABC discovery paradigm. We reproduce Swanson’s discovery of a connection between magnesium and migraine in a novel approach that uses only proteins as the intermediate B terms. We validate our methods by using a cutoff date and evaluate them by calculating precision and recall. In addition to magnesium, we have identified valproic acid and nitric oxide as chemicals which developed links to migraine. We hypothesize, based on protein annotations, that zinc and retinoic acid may play a role in migraine. The ChemoText repository has promise as a data source for drug discovery. PMID:20348023

  1. Integrative DNA, RNA, and Protein Evidence Connects TREML4 to Coronary Artery Calcification

    PubMed Central

    Sen, Shurjo K.; Boelte, Kimberly C.; Barb, Jennifer J.; Joehanes, Roby; Zhao, XiaoQing; Cheng, Qi; Adams, Lila; Teer, Jamie K.; Accame, David S.; Chowdhury, Soma; Singh, Larry N.; Kavousi, Maryam; Peyser, Patricia A.; Quigley, Laura; Priel, Debra Long; Lau, Karen; Kuhns, Douglas B.; Yoshimura, Teizo; Johnson, Andrew D.; Hwang, Shih-Jen; Chen, Marcus Y.; Arai, Andrew E.; Green, Eric D.; Mullikin, James C.; Kolodgie, Frank D.; O’Donnell, Christopher J.; Virmani, Renu; Munson, Peter J.; McVicar, Daniel W.; Biesecker, Leslie G.

    2014-01-01

    Coronary artery calcification (CAC) is a heritable and definitive morphologic marker of atherosclerosis that strongly predicts risk for future cardiovascular events. To search for genes involved in CAC, we used an integrative transcriptomic, genomic, and protein expression strategy by using next-generation DNA sequencing in the discovery phase with follow-up studies using traditional molecular biology and histopathology techniques. RNA sequencing of peripheral blood from a discovery set of CAC cases and controls was used to identify dysregulated genes, which were validated by ClinSeq and Framingham Heart Study data. Only a single gene, TREML4, was upregulated in CAC cases in both studies. Further examination showed that rs2803496 was a TREML4 cis-eQTL and that the minor allele at this locus conferred up to a 6.5-fold increased relative risk of CAC. We characterized human TREML4 and demonstrated by immunohistochemical techniques that it is localized in macrophages surrounding the necrotic core of coronary plaques complicated by calcification (but not in arteries with less advanced disease). Finally, we determined by von Kossa staining that TREML4 colocalizes with areas of microcalcification within coronary plaques. Overall, we present integrative RNA, DNA, and protein evidence implicating TREML4 in coronary artery calcification. Our findings connect multimodal genomics data with a commonly used clinical marker of cardiovascular disease. PMID:24975946

  2. The identification of a heat-shock protein complex in chloroplasts of barley leaves.

    PubMed

    Clarke, A K; Critchley, C

    1992-12-01

    In vivo radiolabeling of chloroplast proteins in barley (Hordeum vulgare L. cv Corvette) leaves and their separation by one-dimensional electrophoresis revealed at least seven heat-shock proteins between 24 and 94 kD, of which most have not been previously identified in this C(3) species. Fractionation into stromal and thylakoid membrane components showed that all chloroplast heat-shock proteins were synthesized on cytoplasmic ribosomes, translocated into the chloroplast, and located in the stroma. Examination of stromal preparations by native (nondissociating) polyacrylamide gel electrophoresis revealed the presence of a high-molecular mass heat-shock protein complex in barley. This complex was estimated to be 250 to 265 kD in size. Dissociation by denaturing polyacrylamide gel electrophoresis revealed a single protein component, a 32-kD heat-shock protein. The synthesis of this protein and the formation of the heat-shock protein complex were dependent on functional cytoplasmic ribosomes. Immunological studies showed that the heat-shock protein complex did not contain any proteins homologous to the alpha-subunit of ribulose bisphosphate carboxylase oxygenase subunit-binding protein. Other features about the complex included the absence of nucleic acid (RNA or DNA) and its nondissociation in the presence of Mg(2+)/ATP. These results suggest that the heat-shock protein complex in barley chloroplasts is a homogeneous octamer of 32-kD subunits. PMID:16653243

  3. The Identification of a Heat-Shock Protein Complex in Chloroplasts of Barley Leaves 1

    PubMed Central

    Clarke, Adrian K.; Critchley, Christa

    1992-01-01

    In vivo radiolabeling of chloroplast proteins in barley (Hordeum vulgare L. cv Corvette) leaves and their separation by one-dimensional electrophoresis revealed at least seven heat-shock proteins between 24 and 94 kD, of which most have not been previously identified in this C3 species. Fractionation into stromal and thylakoid membrane components showed that all chloroplast heat-shock proteins were synthesized on cytoplasmic ribosomes, translocated into the chloroplast, and located in the stroma. Examination of stromal preparations by native (nondissociating) polyacrylamide gel electrophoresis revealed the presence of a high-molecular mass heat-shock protein complex in barley. This complex was estimated to be 250 to 265 kD in size. Dissociation by denaturing polyacrylamide gel electrophoresis revealed a single protein component, a 32-kD heat-shock protein. The synthesis of this protein and the formation of the heat-shock protein complex were dependent on functional cytoplasmic ribosomes. Immunological studies showed that the heat-shock protein complex did not contain any proteins homologous to the α-subunit of ribulose bisphosphate carboxylase oxygenase subunit-binding protein. Other features about the complex included the absence of nucleic acid (RNA or DNA) and its nondissociation in the presence of Mg2+/ATP. These results suggest that the heat-shock protein complex in barley chloroplasts is a homogeneous octamer of 32-kD subunits. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 PMID:16653243

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

    PubMed Central

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

    2013-01-01

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

  5. 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. PMID:23268931

  6. PRISM: a web server and repository for prediction of protein-protein interactions and modeling their 3D complexes.

    PubMed

    Baspinar, Alper; Cukuroglu, Engin; Nussinov, Ruth; Keskin, Ozlem; Gursoy, Attila

    2014-07-01

    The PRISM web server enables fast and accurate prediction of protein-protein interactions (PPIs). The prediction algorithm is knowledge-based. It combines structural similarity and accounts for evolutionary conservation in the template interfaces. The predicted models are stored in its repository. Given two protein structures, PRISM will provide a structural model of their complex if a matching template interface is available. Users can download the complex structure, retrieve the interface residues and visualize the complex model. The PRISM web server is user friendly, free and open to all users at http://cosbi.ku.edu.tr/prism. PMID:24829450

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

    PubMed Central

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

    2014-01-01

    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. PMID:24385580

  8. 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. PMID:26246495

  9. When is an ecological network complex? Connectance drives degree distribution and emerging network properties

    PubMed Central

    Gravel, Dominique

    2014-01-01

    Connectance and degree distributions are important components of the structure of ecological networks. In this contribution, we use a statistical argument and simple network generating models to show that properties of the degree distribution are driven by network connectance. We discuss the consequences of this finding for (1) the generation of random networks in null-model analyses, and (2) the interpretation of network structure and ecosystem properties in relationship with degree distribution. PMID:24688835

  10. 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

  11. 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.

  12. 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.

  13. Characterizing informative sequence descriptors and predicting binding affinities of heterodimeric protein complexes

    PubMed Central

    2015-01-01

    Background Protein-protein interactions (PPIs) are involved in various biological processes, and underlying mechanism of the interactions plays a crucial role in therapeutics and protein engineering. Most machine learning approaches have been developed for predicting the binding affinity of protein-protein complexes based on structure and functional information. This work aims to predict the binding affinity of heterodimeric protein complexes from sequences only. Results This work proposes a support vector machine (SVM) based binding affinity classifier, called SVM-BAC, to classify heterodimeric protein complexes based on the prediction of their binding affinity. SVM-BAC identified 14 of 580 sequence descriptors (physicochemical, energetic and conformational properties of the 20 amino acids) to classify 216 heterodimeric protein complexes into low and high binding affinity. SVM-BAC yielded the training accuracy, sensitivity, specificity, AUC and test accuracy of 85.80%, 0.89, 0.83, 0.86 and 83.33%, respectively, better than existing machine learning algorithms. The 14 features and support vector regression were further used to estimate the binding affinities (Pkd) of 200 heterodimeric protein complexes. Prediction performance of a Jackknife test was the correlation coefficient of 0.34 and mean absolute error of 1.4. We further analyze three informative physicochemical properties according to their contribution to prediction performance. Results reveal that the following properties are effective in predicting the binding affinity of heterodimeric protein complexes: apparent partition energy based on buried molar fractions, relations between chemical structure and biological activity in principal component analysis IV, and normalized frequency of beta turn. Conclusions The proposed sequence-based prediction method SVM-BAC uses an optimal feature selection method to identify 14 informative features to classify and predict binding affinity of heterodimeric protein

  14. 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. PMID:26517730

  15. 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

  16. 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. PMID:24334732

  17. Responses of two protein-protein complexes to solvent stress: does water play a role at the interface?

    PubMed Central

    Kornblatt, J A; Kornblatt, M J; Hoa, G H; Mauk, A G

    1993-01-01

    We have analyzed the stability of the cytochrome c-cytochrome b5 and cytochrome c-cytochrome c oxidase complexes as a function of solvent stress. High concentrations of glycerol were used to displace the two equilibria. Glycerol promotes complex formation between cytochrome c and cytochrome b5 but inhibits that between cytochrome c and cytochrome c oxidase. The results with cytochrome b5 and cytochrome c were expected; the association of this complex is largely entropy driven. Our interpretation is that the cytochrome c-cytochrome b5 complex excludes water. The results with the cytochrome c oxidase and cytochrome c couple were not expected. We interpret them to mean that either glycerol is binding to the oxidase, thereby displacing the cytochrome c, or that water is required at this protein-protein interface. A requirement for substantial quantities of water at the interface of some protein complexes is logical but has been reported only once. PMID:8241386

  18. Isolation of Human Mitotic Protein Phosphatase Complexes: Identification of a Complex between Protein Phosphatase 1 and the RNA Helicase Ddx21

    PubMed Central

    De Wever, Veerle; Lloyd, David C.; Nasa, Isha; Nimick, Mhairi; Trinkle-Mulcahy, Laura; Gourlay, Robert; Morrice, Nick; Moorhead, Greg B. G.

    2012-01-01

    Metazoan mitosis requires remodelling of sub-cellular structures to ensure proper division of cellular and genetic material. Faults often lead to genomic instability, cell cycle arrests and disease onset. These key structural changes are under tight spatial-temporal and post-translational control, with crucial roles for reversible protein phosphorylation. The phosphoprotein phosphatases PP1 and PP2A are paramount for the timely execution of mitotic entry and exit but their interaction partners and substrates are still largely unresolved. High throughput, mass-spectrometry based studies have limited sensitivity for the detection of low-abundance and transient complexes, a typical feature of many protein phosphatase complexes. Moreover, the limited timeframe during which mitosis takes place reduces the likelihood of identifying mitotic phosphatase complexes in asynchronous cells. Hence, numerous mitotic protein phosphatase complexes still await identification. Here we present a strategy to enrich and identify serine/threonine protein phosphatase complexes at the mitotic spindle. We thus identified a nucleolar RNA helicase, Ddx21/Gu, as a novel, direct PP1 interactor. Furthermore, our results place PP1 within the toposome, a Topoisomerase II alpha (TOPOIIα) containing complex with a key role in mitotic chromatin regulation and cell cycle progression, possibly via regulated protein phosphorylation. This study provides a strategy for the identification of further mitotic PP1 partners and the unravelling of PP1 functions during mitosis. PMID:22761809

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

    PubMed

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

    2016-01-27

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

  20. 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.)

  1. Another Role of Proline: Stabilization Interactions in Proteins and Protein Complexes Concerning Proline and Tryptophane

    SciTech Connect

    Biedermannova, Lada; Riley, Kevin E.; Berka, Karel; Hobza, Pavel; Vondrasek, Jiri

    2008-09-11

    Proline–tryptophan complexes derived from experimental structures are investigated by quantum chemical procedures known to properly describe the London dispersion energy. We study two geometrical arrangements: the “L-shaped”, stabilized by an H-bond, and the “stacked-like”, where the two residues are in parallel orientation without any H-bond. Interestingly, the interaction energies in both cases are comparable and very large (~7 kcal mol⁻¹). The strength of stabilization in the stacked arrangement is rather surprising considering the fact that only one partner has an aromatic character. The interaction energy decomposition using the SAPT method further demonstrates the very important role of dispersion energy in such arrangement. To elucidate the structural features responsible for this unexpectedly large stabilization we examined the role of the nitrogen heteroatom and the importance of the cyclicity of the proline residue. We show that the electrostatic interaction due to the presence of the dipole, caused by the nitrogen heteroatom, contributes largely to the strength of the interaction. Nevertheless, the cyclic arrangement of proline, which allows for the largest amount of dispersive contact with the aromatic partner, also has a notable-effect. Geometry optimizations carried out for the “stackedlike” complexes show that the arrangements derived from protein structure are close to their gas phase optimum geometry, suggesting that the environment has only a minor effect on the geometry of the interaction. We conclude that the strength of proline non-covalent interactions, combined with this residue’s rigidity, might be the explanation for its prominent role in protein stabilization and recognition processes.

  2. 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.

  3. MICAL-Family Proteins: Complex Regulators of the Actin Cytoskeleton

    PubMed Central

    Giridharan, Sai Srinivas Panapakkam

    2014-01-01

    Abstract Significance: The molecules interacting with CasL (MICAL) family members participate in a multitude of activities, including axonal growth cone repulsion, membrane trafficking, apoptosis, and bristle development in flies. An interesting feature of MICAL proteins is the presence of an N-terminal flavo-mono-oxygenase domain. This mono-oxygenase domain generates redox potential with which MICALs can either oxidize proteins or produce reactive oxygen species (ROS). Actin is one such protein that is affected by MICAL function, leading to dramatic cytoskeletal rearrangements. This review describes the MICAL-family members, and discusses their mechanisms of actin-binding and regulation of actin cytoskeleton organization. Recent Advances: Recent studies show that MICALs directly induce oxidation of actin molecules, leading to actin depolymerization. ROS production by MICALs also causes oxidation of collapsin response mediator protein-2, a microtubule assembly promoter, which subsequently undergoes phosphorylation. Critical Issues: MICAL proteins oxidize proteins through two mechanisms: either directly by oxidizing methionine residues or indirectly via the production of ROS. It remains unclear whether MICAL proteins employ both mechanisms or whether the activity of MICAL-family proteins might vary with different substrates. Future Directions: The identification of additional substrates oxidized by MICAL will shed new light on MICAL protein function. Additional directions include expanding studies toward the MICAL-like homologs that lack flavin adenine dinucleotide domains and oxidation activity. Antioxid. Redox Signal. 20, 2059–2073. PMID:23834433

  4. Ahnak protein activates protein kinase C (PKC) through dissociation of the PKC-protein phosphatase 2A complex.

    PubMed

    Lee, In Hye; Lim, Hee Jung; Yoon, Suhyeon; Seong, Je Kyung; Bae, Duk Soo; Rhee, Sue Goo; Bae, Yun Soo

    2008-03-01

    We have previously reported that central repeated units (CRUs) of Ahnak act as a scaffolding protein networking phospholipase Cgamma and protein kinase C (PKC). Here, we demonstrate that an Ahnak derivative consisting of four central repeated units binds and activates PKC-alpha in a phosphatidylserine/1,2-dioleoyl-sn-glycerol-independent manner. Moreover, NIH3T3 cells expressing the 4 CRUs of Ahnak showed enhanced c-Raf, MEK, and Erk phosphorylation in response to phorbol 12-myristate 13-acetate (PMA) compared with parental cells. To evaluate the effect of loss-of-function of Ahnak in cell signaling, we investigated PKC activation and Raf phosphorylation in embryonic fibroblast cells (MEFs) of the Ahnak knock-out (Ahnak(-/-)) mouse. Membrane translocation of PKC-alpha and phosphorylation of Raf in response to PMA or platelet-derived growth factor were decreased in Ahnak null MEF cells compared with wild type MEFs. Several lines of evidence suggest that PKC-alpha activity is regulated through association with protein phosphatase 2A (PP2A). A co-immunoprecipitation assay indicated that the association of PKC-alpha with PP2A was disrupted in NIH3T3 cells expressing 4 CRUs of Ahnak in response to PMA. Consistently, Ahnak null MEF cells stimulated by PMA showed enhanced PKC-PP2A complex formation, and add-back expression of Ahnak into Ahnak null MEF cells abolished the PKC-PP2A complex formation in response to PMA. These data indicate that Ahnak potentiates PKC activation through inhibiting the interaction of PKC with PP2A. PMID:18174170

  5. The electron conduction of photosynthetic protein complexes embedded in a membrane.

    PubMed

    Stamouli, A; Frenken, J W M; Oosterkamp, T H; Cogdell, R J; Aartsma, T J

    2004-02-27

    The conductivity of two photosynthetic protein-pigment complexes, a light harvesting 2 complex and a reaction center, was measured with an atomic force microscope capable of performing electrical measurements. Current-voltage measurements were performed on complexes embedded in their natural environment. Embedding the complexes in lipid bilayers made it possible to discuss the different conduction behaviors of the two complexes in light of their atomic structure. PMID:14988007

  6. 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.

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

    NASA Astrophysics Data System (ADS)

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

    2015-10-01

    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.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. Electronic supplementary information (ESI) available. See DOI: 10.1039/c5nr05147a

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

    PubMed

    Kovács, Krisztián A; Steinmann, Myriam; Halfon, Olivier; Magistretti, Pierre J; Cardinaux, Jean-René

    2015-11-01

    CREB-binding protein (CBP) and p300 are transcriptional coactivators involved in numerous biological processes that affect cell growth, transformation, differentiation, and development. In this study, we provide evidence of the involvement of homeodomain-interacting protein kinase 2 (HIPK2) in the regulation of CBP activity. We show that HIPK2 interacts with and phosphorylates several regions of CBP. We demonstrate that serines 2361, 2363, 2371, 2376, and 2381 are responsible for the HIPK2-induced mobility shift of CBP C-terminal activation domain. Moreover, we show that HIPK2 strongly potentiates the transcriptional activity of CBP. However, our data suggest that HIPK2 activates CBP mainly by counteracting the repressive action of cell cycle regulatory domain 1 (CRD1), located between amino acids 977 and 1076, independently of CBP phosphorylation. Our findings thus highlight a complex regulation of CBP activity by HIPK2, which might be relevant for the control of specific sets of target genes involved in cellular proliferation, differentiation and apoptosis. PMID:26247811

  9. 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

  10. 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

  11. Towards a Hierarchical Strategy to Explore Multi-Scale IP/MS Data for Protein Complexes

    PubMed Central

    Kutzera, Joachim; Smilde, Age K.; Wilderjans, Tom F.; Hoefsloot, Huub C. J.

    2015-01-01

    Protein interaction in cells can be described at different levels. At a low interaction level, proteins function together in small, stable complexes and at a higher level, in sets of interacting complexes. All interaction levels are crucial for the living organism, and one of the challenges in proteomics is to measure the proteins at their different interaction levels. One common method for such measurements is immunoprecipitation followed by mass spectrometry (IP/MS), which has the potential to probe the different protein interaction forms. However, IP/MS data are complex because proteins, in their diverse interaction forms, manifest themselves in different ways in the data. Numerous bioinformatic tools for finding protein complexes in IP/MS data are currently available, but most tools do not provide information about the interaction level of the discovered complexes, and no tool is geared specifically to unraveling and visualizing these different levels. We present a new bioinformatic tool to explore IP/MS datasets for protein complexes at different interaction levels and show its performance on several real–life datasets. Our tool creates clusters that represent protein complexes, but unlike previous methods, it arranges them in a tree–shaped structure, reporting why specific proteins are predicted to build a complex and where it can be divided into smaller complexes. In every data analysis method, parameters have to be chosen. Our method can suggest values for its parameters and comes with adapted visualization tools that display the effect of the parameters on the result. The tools provide fast graphical feedback and allow the user to interact with the data by changing the parameters and examining the result. The tools also allow for exploring the different organizational levels of the protein complexes in a given dataset. Our method is available as GNU-R source code and includes examples at www.bdagroup.nl. PMID:26448546

  12. 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.

  13. Immobilization of two organometallic complexes into a single cage to construct protein-based microcompartments.

    PubMed

    Maity, Basudev; Fukumori, Kazuki; Abe, Satoshi; Ueno, Takafumi

    2016-04-01

    Natural protein-based microcompartments containing multiple enzymes promote cascade reactions within cells. We use the apo-ferritin protein cage to mimic such biocompartments by immobilizing two organometallic Ir and Pd complexes into the single protein cage. Precise locations of the metals and their accumulation mechanism were studied by X-ray crystallography. PMID:27021005

  14. A complex of three related membrane proteins is conserved on malarial merozoites

    PubMed Central

    Rayavara, Kempaiah; Rajapandi, Thavamani; Wollenberg, Kurt; Kabat, Juraj; Fischer, Elizabeth R.; Desai, Sanjay A.

    2009-01-01

    Invasion of human red blood cells by the malaria parasite P. falciparum is a coordinated, multi-step process. Here, we describe three novel integral membrane proteins that colocalize on the inner membrane complex immediately beneath the merozoite plasma membrane. Each has 6 predicted transmembrane domains and is conserved in diverse apicomplexan parasites. Immunoprecipitation studies using specific antibodies reveal that these proteins assemble into a heteromeric complex. Each protein was also expressed on insect cells using the baculovirus vector system with a truncated SUMO tag that facilitates maximal expression and protein purification while permitting cleavage with SUMO protease to release unmodified parasite protein. The expressed proteins were successfully reconstituted into artificial liposomes, but were not recognized by human immune sera. Because all three genes are highly conserved in apicomplexan parasites, the complex formed by their encoded proteins likely serves an essential role for invasive merozoites. PMID:19465059

  15. Fractionation of Thylakoid Membranes with the Nonionic Detergent Octyl-beta-d-glucopyranoside: RESOLUTION OF CHLOROPHYLL-PROTEIN COMPLEX II INTO TWO CHLOROPHYLL-PROTEIN COMPLEXES.

    PubMed

    Camm, E L; Green, B R

    1980-09-01

    The detergent octyl-beta-d-glucopyranoside (30 millimolar in 2 millimolar Tris-maleate, pH 7.0) preferentially extracts complexes containing protein and chlorophylls a plus b (CP) from spinach, leaving a residue highly enriched in CP I (P700-chlorophyll a protein). Use of the detergent results in a relatively gentle extraction since little free chlorophyll is formed and since sodium dodecyl sulfate-gel electrophoresis (on 10% acrylamide) of the extract also reveals the presence of two minor chlorophyll a complexes (apparent molecular weight, 47,000 and 43,000) instead of the usual single complex. The major complex preserved is CP 64, a chlorophyll a/b complex (apparent molecular weight, 64,000) which is an oligomer of another chlorophyll a/b complex, CP 27, the light-harvesting complex (apparent molecular weight, 27,000). Dissociation of each complex reveals two polypeptides (molecular weight, 32,000 and 28,000) and limited proteolysis confirms that those of CP 64 have the same structure as those of CP 27. An additional chlorophyll a/b complex (apparent molecular weight, 29,000) is clearly separable from CP 27, and differs from it and CP 64 in having a higher chlorophyll a/b ratio and a single polypeptide (molecular weight, 29,000) which differs structurally from those of the other complexes. PMID:16661449

  16. 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. PMID:26873405

  17. 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. PMID:15664347

  18. In vivo protein complex topologies: Sights through a cross-linking lens

    PubMed Central

    Bruce, James E.

    2013-01-01

    Proteins are a remarkable class of molecules that exhibit wide diversity of shapes or topological features that underpin protein interactions and give rise to biological function. In addition to quantitation of abundance levels of proteins in biological systems under a variety of conditions, the field of proteome research has as a primary mission the assignment of function for proteins and if possible, illumination of factors that enable function. For many years, chemical cross-linking methods have been used to provide structural data on single purified proteins and purified protein complexes. However, these methods also offer the alluring possibility to extend capabilities to complex biological samples such as cell lysates or intact living cells where proteins may exhibit native topological features that do not exist in purified form. Recent efforts are beginning to provide glimpses of protein complexes and topologies in cells that suggest continued development will yield novel capabilities to view functional topological features of many protein and complexes as they exist in cells, tissues or other complex samples. This review will describe rationale, challenges and a few success stories along the path of development of cross-linking technologies for measurement of in vivo protein interaction topologies. PMID:22610688

  19. An updated version of NPIDB includes new classifications of DNA-protein complexes and their families.

    PubMed

    Zanegina, Olga; Kirsanov, Dmitriy; Baulin, Eugene; Karyagina, Anna; Alexeevski, Andrei; Spirin, Sergey

    2016-01-01

    The recent upgrade of nucleic acid-protein interaction database (NPIDB, http://npidb.belozersky.msu.ru/) includes a newly elaborated classification of complexes of protein domains with double-stranded DNA and a classification of families of related complexes. Our classifications are based on contacting structural elements of both DNA: the major groove, the minor groove and the backbone; and protein: helices, beta-strands and unstructured segments. We took into account both hydrogen bonds and hydrophobic interaction. The analyzed material contains 1942 structures of protein domains from 748 PDB entries. We have identified 97 interaction modes of individual protein domain-DNA complexes and 17 DNA-protein interaction classes of protein domain families. We analyzed the sources of diversity of DNA-protein interaction modes in different complexes of one protein domain family. The observed interaction mode is sometimes influenced by artifacts of crystallization or diversity in secondary structure assignment. The interaction classes of domain families are more stable and thus possess more biological sense than a classification of single complexes. Integration of the classification into NPIDB allows the user to browse the database according to the interacting structural elements of DNA and protein molecules. For each family, we present average DNA shape parameters in contact zones with domains of the family. PMID:26656949

  20. Evidence for a structural role for chlorophyll in chlorophyll-protein complexes.

    PubMed

    Jennings, R C; Garlaschi, F M; Forti, G; Gerola, P D

    1979-11-23

    1. Chymotrypsin treatment of spinach chloroplast membranes does not change the electrophoretic mobility of either chlorophyll-protein complex 1 or 2. 2. The extraction of lipids with 80% acetone after treatment of the membranes with chymotrypsin reveals that the polypeptide components of both chlorophyll-protein complexes had been extensively digested. The extraction of carotenes with petroleum ether under the same conditions does not change the electrophoretic mobility of the chlorophyll-protein complexes. 3. Fluorescence polarisation studies of chlorophyll-protein complex 2 reveal that the chymotrypsin digestion of this complex does not result in changes of mutual orientation or distance apart of chlorophyll a, chlorophyll b or carotenoid. 4. Two polypeptide components have been detected after lipid extraction of electrophoretically purified chlorophyll-protein complexes 1 and 2. The SDS molecular weights are 24 000 and 27 000 for complex 2, and 68 000 and 64 000 for complex 1. 5. We conclude that chlorophyll performs an important structural function in both chlorophyll-protein complexes. PMID:508798

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

    PubMed

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

    2015-11-01

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

  2. A Three-Hybrid System to Probe In Vivo Protein-Protein Interactions: Application to the Essential Proteins of the RD1 Complex of M. tuberculosis

    PubMed Central

    Bhalla, Kuhulika; Ghosh, Anamika; Kumar, Krishan; Kumar, Sushil; Ranganathan, Anand

    2011-01-01

    Background Protein-protein interactions play a crucial role in enabling a pathogen to survive within a host. In many cases the interactions involve a complex of proteins rather than just two given proteins. This is especially true for pathogens like M. tuberculosis that are able to successfully survive the inhospitable environment of the macrophage. Studying such interactions in detail may help in developing small molecules that either disrupt or augment the interactions. Here, we describe the development of an E. coli based bacterial three-hybrid system that can be used effectively to study ternary protein complexes. Methodology/Principal Findings The protein-protein interactions involved in M. tuberculosis pathogenesis have been used as a model for the validation of the three-hybrid system. Using the M. tuberculosis RD1 encoded proteins CFP10, ESAT6 and Rv3871 for our proof-of-concept studies, we show that the interaction between the proteins CFP10 and Rv3871 is strengthened and stabilized in the presence of ESAT6, the known heterodimeric partner of CFP10. Isolating peptide candidates that can disrupt crucial protein-protein interactions is another application that the system offers. We demonstrate this by using CFP10 protein as a disruptor of a previously established interaction between ESAT6 and a small peptide HCL1; at the same time we also show that CFP10 is not able to disrupt the strong interaction between ESAT6 and another peptide SL3. Conclusions/Significance The validation of the three-hybrid system paves the way for finding new peptides that are stronger binders of ESAT6 compared even to its natural partner CFP10. Additionally, we believe that the system offers an opportunity to study tri-protein complexes and also perform a screening of protein/peptide binders to known interacting proteins so as to elucidate novel tri-protein complexes. PMID:22087330

  3. Multiple functions of Kip-related protein5 connect endoreduplication and cell elongation.

    PubMed

    Jégu, Teddy; Latrasse, David; Delarue, Marianne; Mazubert, Christelle; Bourge, Mickaël; Hudik, Elodie; Blanchet, Sophie; Soler, Marie-Noëlle; Charon, Céline; De Veylder, Lieven; Raynaud, Cécile; Bergounioux, Catherine; Benhamed, Moussa

    2013-04-01

    Despite considerable progress in our knowledge regarding the cell cycle inhibitor of the Kip-related protein (KRP) family in plants, less is known about the coordination of endoreduplication and cell differentiation. In animals, the role of cyclin-dependent kinase (CDK) inhibitors as multifunctional factors coordinating cell cycle regulation and cell differentiation is well documented and involves not only the inhibition of CDK/cyclin complexes but also other mechanisms, among them the regulation of transcription. Interestingly, several plant KRPs have a punctuated distribution in the nucleus, suggesting that they are associated with heterochromatin. Here, one of these chromatin-bound KRPs, KRP5, has been studied in Arabidopsis (Arabidopsis thaliana). KRP5 is expressed in endoreduplicating cells, and loss of KRP5 function decreases endoreduplication, indicating that KRP5 is a positive regulator of endoreduplication. This regulation relies on several mechanisms: in addition to its role in cyclin/CDK kinase inhibition previously described, chromatin immunoprecipitation sequencing data combined with transcript quantification provide evidence that KRP5 regulates the transcription of genes involved in cell wall organization. Furthermore, KRP5 overexpression increases chromocenter decondensation and endoreduplication in the Arabidopsis trithorax-related protein5 (atxr5) atxr6 double mutant, which is deficient for the deposition of heterochromatin marks. Hence, KRP5 could bind chromatin to coordinately control endoreduplication and chromatin structure and allow the expression of genes required for cell elongation. PMID:23426196

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

    PubMed

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

    2007-08-01

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

  5. 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

  6. Domain stealing by receptors in a protein transport complex.

    PubMed

    Hulett, Joanne M; Walsh, Peter; Lithgow, Trevor

    2007-09-01

    The mitochondrion is an essential cellular compartment in eukaryotes. The mitochondrial proteins Tom20 and Tom22 are receptors that ensure recognition and binding of proteins imported for mitochondrial biogenesis. Comparison of the sequence for the Tom20 and Tom22 subunits in the yeasts Saccharomyces cerevisiae and Saccharomyces castellii, show a rare case of domain stealing, where in Saccharomyces castellii Tom22 has lost an acidic domain, and Tom20 has gained one. This example of domain stealing is a snapshot of evolution in action and provides excellent evidence that Tom20 and Tom22 are subunits of a single, composite receptor that binds precursor proteins for import into mitochondria. PMID:17586602

  7. 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. PMID:26712034

  8. 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

  9. Mapping the Protein Interaction Network for TFIIB-Related Factor Brf1 in the RNA Polymerase III Preinitiation Complex

    PubMed Central

    Khoo, Seok-Kooi; Wu, Chih-Chien; Lin, Yu-Chun; Lee, Jin-Cheng

    2014-01-01

    TFIIB-related factor Brf1 is essential for RNA polymerase (Pol) III recruitment and open-promoter formation in transcription initiation. We site specifically incorporated a nonnatural amino acid cross-linker into Brf1 to map its protein interaction targets in the preinitiation complex (PIC). Our cross-linking analysis in the N-terminal domain of Brf1 indicated a pattern of multiple protein interactions reminiscent of TFIIB in the Pol active-site cleft. In addition to the TFIIB-like protein interactions, the Brf1 cyclin repeat subdomain is in contact with the Pol III-specific C34 subunit. With site-directed hydroxyl radical probing, we further revealed the binding between Brf1 cyclin repeats and the highly conserved region connecting C34 winged-helix domains 2 and 3. In contrast to the N-terminal domain of Brf1, the C-terminal domain contains extensive binding sites for TBP and Bdp1 to hold together the TFIIIB complex on the promoter. Overall, the domain architecture of the PIC derived from our cross-linking data explains how individual structural subdomains of Brf1 integrate the protein network from the Pol III active center to the promoter for transcription initiation. PMID:24277937

  10. Mapping the protein interaction network for TFIIB-related factor Brf1 in the RNA polymerase III preinitiation complex.

    PubMed

    Khoo, Seok-Kooi; Wu, Chih-Chien; Lin, Yu-Chun; Lee, Jin-Cheng; Chen, Hung-Ta

    2014-02-01

    TFIIB-related factor Brf1 is essential for RNA polymerase (Pol) III recruitment and open-promoter formation in transcription initiation. We site specifically incorporated a nonnatural amino acid cross-linker into Brf1 to map its protein interaction targets in the preinitiation complex (PIC). Our cross-linking analysis in the N-terminal domain of Brf1 indicated a pattern of multiple protein interactions reminiscent of TFIIB in the Pol active-site cleft. In addition to the TFIIB-like protein interactions, the Brf1 cyclin repeat subdomain is in contact with the Pol III-specific C34 subunit. With site-directed hydroxyl radical probing, we further revealed the binding between Brf1 cyclin repeats and the highly conserved region connecting C34 winged-helix domains 2 and 3. In contrast to the N-terminal domain of Brf1, the C-terminal domain contains extensive binding sites for TBP and Bdp1 to hold together the TFIIIB complex on the promoter. Overall, the domain architecture of the PIC derived from our cross-linking data explains how individual structural subdomains of Brf1 integrate the protein network from the Pol III active center to the promoter for transcription initiation. PMID:24277937

  11. 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.

  12. Single-molecule study of protein-DNA target search mechanisms for dimer-active protein complexes

    NASA Astrophysics Data System (ADS)

    Landry, Markita; Huang, Wai Mun; Chemla, Yann

    2012-02-01

    Protein-DNA interactions are essential to cellular processes, many of which require proteins to recognize a specific DNA target-site. This search process is well-documented for monomeric proteins, but not as well understood for systems that require dimerization at the target site for activity. We present a single-molecule study of the target-search mechanism of Protelomerase TelK, a recombinase-like protein that is only active as a dimer. We observe that TelK undergoes 1D diffusion on non-target DNA as a monomer, as expected, but becomes immobile on DNA as a dimer or oligomer despite the absence of its target site. We further show that TelK condenses non-target DNA upon dimerization, forming a tightly bound nucleo-protein complex. Together with simulations, our results suggest a search model whereby monomers diffuse along DNA, and subsequently dimerize to form an active complex on target DNA. These results show that target-finding occurs faster than nonspecific dimerization at biologically relevant protein concentrations. This model may provide insights into the search mechanisms of proteins that are active as multimeric complexes for a more accurate and comprehensive model for the target-search process by sequence specific proteins.

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

    PubMed

    Tilley, S David; Francis, Matthew B

    2006-02-01

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

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

    PubMed Central

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

    2015-01-01

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

  15. 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.

  16. 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.

  17. Accessory proteins stabilize the acceptor complex for synaptobrevin, the 1:1 syntaxin/SNAP-25 complex

    PubMed Central

    Weninger, Keith; Bowen, Mark E.; Choi, Ucheor B.; Chu, Steven; Brunger, Axel T.

    2010-01-01

    Summary Formation of a binary complex between syntaxin and SNAP-25 (synaptosome-associated protein of 25 kDa) at the active zone is believed to precede assembly of the ternary SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) complex that is essential for neurotransmitter release. Despite its importance in models of synaptic neurotransmitter release, this binary complex has been difficult to characterize by bulk methods due to the prevalence of a 2:1 dead-end species. Here we used single molecule fluorescence resonance energy transfer (smFRET) to study the structure and dynamics of the 1:1 syntaxin/SNAP-25 binary complex. The binary complex is conformationally variable with FRET efficiency states often changing on the second timescale. One state corresponds to a parallel three-helix bundle configuration, while other states correspond to configurations with one of the SNAP-25 SNARE domains dissociated. All configurations of the binary complex are rapidly locked into the single three-helix bundle configuration by the addition of synaptobrevin. Remarkably, upon addition of complexin, Munc13, Munc18, or synaptotagmin, a similar effect is observed. Thus, the 1:1 binary complex serves as a dynamic acceptor for synaptobrevin binding, and interactions with accessory proteins stabilize this acceptor. In a high protein density cellular environment the syntaxin/SNAP-25 complex is therefore expected to be in the configuration where it can rapidly interact with synaptobrevin so its formation is unlikely a limiting step for SNARE-mediated neurotransmitter release. PMID:18275821

  18. SVP-like MADS-box protein from Carya cathayensis forms higher-order complexes.

    PubMed

    Wang, Jingjing; Hou, Chuanming; Huang, Jianqin; Wang, Zhengjia; Xu, Yingwu

    2015-03-01

    To properly regulate plant flowering time and construct floral pattern, MADS-domain containing transcription factors must form multimers including homo- and hetero-dimers. They are also active in forming hetero-higher-order complexes with three to five different molecules. However, it is not well known if a MADS-box protein can also form homo-higher-order complex. In this study a biochemical approach is utilized to provide insight into the complex formation for an SVP-like MADS-box protein cloned from hickory. The results indicated that the protein is a heterogeneous higher-order complex with the peak population containing over 20 monomers. Y2H verified the protein to form homo-complex in yeast cells. Western blot of the hickory floral bud sample revealed that the protein exists in higher-order polymers in native. Deletion assays indicated that the flexible C-terminal residues are mainly responsible for the higher-order polymer formation and the heterogeneity. Current results provide direct biochemical evidences for an active MADS-box protein to be a high order complex, much higher than a quartermeric polymer. Analysis suggests that a MADS-box subset may be able to self-assemble into large complexes, and thereby differentiate one subfamily from the other in a higher-order structural manner. Present result is a valuable supplement to the action of mechanism for MADS-box proteins in plant development. PMID:25602439

  19. 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

  20. Single-molecule Observation of Protein Folding in Symmetric GroEL-(GroES)2 Complexes*

    PubMed Central

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

    2012-01-01

    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

  1. Mass spectrometry-based shotgun proteomic analysis of C. elegans protein complexes.

    PubMed

    Fonslow, Bryan R; Moresco, James J; Tu, Patricia G; Aalto, Antti P; Pasquinelli, Amy E; Dillin, Andrew G; Yates, John R

    2014-01-01

    Mass spectrometry (MS)-based shotgun proteomics is an enabling technology for the study of C. elegans proteins. When coupled with co-immunoprecipitation (CoIP), new interactions and functions among proteins can be discovered. We provide a general background on protein complexes and methods for their analysis, along with the lifecycle and interaction types of proteins that ultimately define the identifiable components of protein complexes. We highlight traditional biochemical methods to evaluate whether the complexes are sufficiently pure and abundant for analysis with shotgun proteomics. We present two CoIP-MS case studies of protein complexes from C. elegans, using both endogenous and fusion protein antibodies to illustrate the important aspects of their analyses. We discuss results from mass spectrometers with differences in mass accuracy and resolution, along with the relevant information that can be extracted from the data generated, such as protein relative abundance, post-translational modifications, and identification confidence. Finally, we illustrate how comparative analysis can reveal candidate binding partners for biological follow-up and validation. This chapter should act as a complement and extension to the WormBook chapter Biochemistry and molecular biology, which describes tandem affinity purification (TAP) of protein complexes for analysis by mass spectrometry. PMID:24967700

  2. Production of Protein Complexes in Non-methylotrophic and Methylotrophic Yeasts : Nonmethylotrophic and Methylotrophic Yeasts.

    PubMed

    Fernández, Francisco J; López-Estepa, Miguel; Querol-García, Javier; Vega, M Cristina

    2016-01-01

    Protein complexes can be produced in multimilligram quantities using nonmethylotrophic and methylotrophic yeasts such as Saccharomyces cerevisiae and Komagataella (Pichia) pastoris. Yeasts have distinct advantages as hosts for recombinant protein production owing to their cost efficiency, ease of cultivation and genetic manipulation, fast growth rates, capacity to introduce post-translational modifications, and high protein productivity (yield) of correctly folded protein products. Despite those advantages, yeasts have surprisingly lagged behind other eukaryotic hosts in their use for the production of multisubunit complexes. As our knowledge of the metabolic and genomic bottlenecks that yeast microorganisms face when overexpressing foreign proteins expands, new possibilities emerge for successfully engineering yeasts as superb expression hosts. In this chapter, we describe the current state of the art and discuss future possibilities for the development of yeast-based systems for the production of protein complexes. PMID:27165323

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

    PubMed

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

    2008-12-01

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

  4. Reconciling the regulatory role of Munc18 proteins in SNARE-complex assembly

    PubMed Central

    Rehman, Asma; Archbold, Julia K.; Hu, Shu-Hong; Norwood, Suzanne J.; Collins, Brett M.; Martin, Jennifer L.

    2014-01-01

    Membrane fusion is essential for human health, playing a vital role in processes as diverse as neurotransmission and blood glucose control. Two protein families are key: (1) the Sec1p/Munc18 (SM) and (2) the soluble N-ethylmaleimide-sensitive attachment protein receptor (SNARE) proteins. Whilst the essential nature of these proteins is irrefutable, their exact regulatory roles in membrane fusion remain controversial. In particular, whether SM proteins promote and/or inhibit the SNARE-complex formation required for membrane fusion is not resolved. Crystal structures of SM proteins alone and in complex with their cognate SNARE proteins have provided some insight, however, these structures lack the transmembrane spanning regions of the SNARE proteins and may not accurately reflect the native state. Here, we review the literature surrounding the regulatory role of mammalian Munc18 SM proteins required for exocytosis in eukaryotes. Our analysis suggests that the conflicting roles reported for these SM proteins may reflect differences in experimental design. SNARE proteins appear to require C-terminal immobilization or anchoring, for example through a transmembrane domain, to form a functional fusion complex in the presence of Munc18 proteins. PMID:25485130

  5. 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

  6. Comprehensive analysis of heterotrimeric G-protein complex diversity and their interactions with GPCRs in solution

    PubMed Central

    Hillenbrand, Matthias; Schori, Christian; Schöppe, Jendrik; Plückthun, Andreas

    2015-01-01

    Agonist binding to G-protein–coupled receptors (GPCRs) triggers signal transduction cascades involving heterotrimeric G proteins as key players. A major obstacle for drug design is the limited knowledge of conformational changes upon agonist binding, the details of interaction with the different G proteins, and the transmission to movements within the G protein. Although a variety of different GPCR/G protein complex structures would be needed, the transient nature of this complex and the intrinsic instability against dissociation make this endeavor very challenging. We have previously evolved GPCR mutants that display higher stability and retain their interaction with G proteins. We aimed at finding all G-protein combinations that preferentially interact with neurotensin receptor 1 (NTR1) and our stabilized mutants. We first systematically analyzed by coimmunoprecipitation the capability of 120 different G-protein combinations consisting of αi1 or αsL and all possible βγ-dimers to form a heterotrimeric complex. This analysis revealed a surprisingly unrestricted ability of the G-protein subunits to form heterotrimeric complexes, including βγ-dimers previously thought to be nonexistent, except for combinations containing β5. A second screen on coupling preference of all G-protein heterotrimers to NTR1 wild type and a stabilized mutant indicated a preference for those Gαi1βγ combinations containing γ1 and γ11. Heterotrimeric G proteins, including combinations believed to be nonexistent, were purified, and complexes with the GPCR were prepared. Our results shed new light on the combinatorial diversity of G proteins and their coupling to GPCRs and open new approaches to improve the stability of GPCR/G-protein complexes. PMID:25733868

  7. Complex Rotating Waves and Long Transients in a Ring Network of Electrochemical Oscillators with Sparse Random Cross-Connections.

    PubMed

    Sebek, Michael; Tönjes, Ralf; Kiss, István Z

    2016-02-12

    We perform experiments and phase model simulations with a ring network of oscillatory electrochemical reactions to explore the effect of random connections and nonisochronicity of the interactions on the pattern formation. A few additional links facilitate the emergence of the fully synchronized state. With larger nonisochronicity, complex rotating waves or persistent irregular phase dynamics can derail the convergence to global synchronization. The observed long transients of irregular phase dynamics exemplify the possibility of a sudden onset of hypersynchronous behavior without any external stimulus or network reorganization. PMID:26919024

  8. The alternative oxidases: simple oxidoreductase proteins with complex functions.

    PubMed

    Young, Luke; Shiba, Tomoo; Harada, Shigeharu; Kita, Kiyoshi; Albury, Mary S; Moore, Anthony L

    2013-10-01

    The alternative oxidases are membrane-bound monotopic terminal electron transport proteins found in all plants and in some agrochemically important fungi and parasites including Trypansoma brucei, which is the causative agent of trypanosomiasis. They are integral membrane proteins and reduce oxygen to water in a four electron process. The recent elucidation of the crystal structure of the trypanosomal alternative oxidase at 2.85 Å (1 Å=0.1 nm) has revealed salient structural features necessary for its function. In the present review we compare the primary and secondary ligation spheres of the alternative oxidases with other di-iron carboxylate proteins and propose a mechanism for the reduction of oxygen to water. PMID:24059524

  9. 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.

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

    PubMed

    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. PMID:27198202

  11. NMR techniques in drug delivery: application to zein protein complexes.

    PubMed

    Sousa, F F O; Luzardo-Álvarez, Asteria; Blanco-Méndez, José; Martín-Pastor, Manuel

    2012-12-15

    Zein is a protein containing a large amount of nonpolar amino acids, which has shown the ability to form aggregates and entrap solutes, such as drugs and amino acids. NMR techniques were used to detect binding interactions and measure affinity between zein and three different drugs: tetracycline, amoxicillin and indomethacin. The release study of zein microparticle formulations containing any of these drugs was confronted with the affinity results, showing a remarkable correlation. The feasible methodology employed, focused in the functionality of the protein-drug interaction, can be very promising for the rational design of appropriate drug vehicles for drug delivery. PMID:23041651

  12. Determining exon connectivity in complex mRNAs by nanopore sequencing.

    PubMed

    Bolisetty, Mohan T; Rajadinakaran, Gopinath; Graveley, Brenton R

    2015-01-01

    Short-read high-throughput RNA sequencing, though powerful, is limited in its ability to directly measure exon connectivity in mRNAs that contain multiple alternative exons located farther apart than the maximum read length. Here, we use the Oxford Nanopore MinION sequencer to identify 7,899 'full-length' isoforms expressed from four Drosophila genes, Dscam1, MRP, Mhc, and Rdl. These results demonstrate that nanopore sequencing can be used to deconvolute individual isoforms and that it has the potential to be a powerful method for comprehensive transcriptome characterization. PMID:26420219

  13. 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. PMID:27235398

  14. 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.

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

    PubMed

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

    1999-08-01

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

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

    PubMed

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

    2013-03-01

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

  17. A cryptobiosis-specific 19S protein complex of Artemia salina gastrulae.

    PubMed

    De Herdt, E; De Voeght, F; Clauwaert, J; Kondo, M; Slegers, H

    1981-01-15

    The postribosomal supernatant of Artemia salina cryptobiotic embryos contains a large quantity of a 19S protein complex. An amount of 3.6 mg/g of cysts is measured by immunoprecipitation with anti-(19S protein complex) antibody. The quantity of this complex decreases during further development to nauplius larvae to only 15% of the quantity present in cryptobiotic embryos. The complex was no longer detectable after 7 days of growth. The 27000-Mr protein subunit of the 19S complex is not synthesized by mRNA isolated from cryptobiotic embryos. The cryptobiosis-specific complex has Mr 573000 and 610000 as calculated from light-scattering and sedimentation-diffusion measurements respectively. The 19S homocomplex contains 20-23 27000-Mr proteins and has no function in the translation of homologous mRNA. From hydrodynamic data a hydration of 1.25 g of water/g of protein is calculated. The abundant presence of the 19S protein complex in cryptobiotic embryos and the absence of synthesis during development to nauplius larvae indicate a functional role during the cryptobiotic process in early embryogenesis. A role in maintaining the water content of the cytoplasm above a critical threshold during desiccation is suggested. PMID:7305995

  18. Structural Analysis of Protein-RNA Complexes in Solution Using NMR Paramagnetic Relaxation Enhancements.

    PubMed

    Hennig, Janosch; Warner, Lisa R; Simon, Bernd; Geerlof, Arie; Mackereth, Cameron D; Sattler, Michael

    2015-01-01

    Biological activity in the cell is predominantly mediated by large multiprotein and protein-nucleic acid complexes that act together to ensure functional fidelity. Nuclear magnetic resonance (NMR) spectroscopy is the only method that can provide information for high-resolution three-dimensional structures and the conformational dynamics of these complexes in solution. Mapping of binding interfaces and molecular interactions along with the characterization of conformational dynamics is possible for very large protein complexes. In contrast, de novo structure determination by NMR becomes very time consuming and difficult for protein complexes larger than 30 kDa as data are noisy and sparse. Fortunately, high-resolution structures are often available for individual domains or subunits of a protein complex and thus sparse data can be used to define their arrangement and dynamics within the assembled complex. In these cases, NMR can therefore be efficiently combined with complementary solution techniques, such as small-angle X-ray or neutron scattering, to provide a comprehensive description of the structure and dynamics of protein complexes in solution. Particularly useful are NMR-derived paramagnetic relaxation enhancements (PREs), which provide long-range distance restraints (ca. 20Å) for structural analysis of large complexes and also report on conformational dynamics in solution. Here, we describe the use of PREs from sample production to structure calculation, focusing on protein-RNA complexes. On the basis of recent examples from our own research, we demonstrate the utility, present protocols, and discuss potential pitfalls when using PREs for studying the structure and dynamic features of protein-RNA complexes. PMID:26068746

  19. Remotely Sensed Image Classification by Complex Network Eigenvalue and Connected Degree

    PubMed Central

    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. PMID:22242041

  20. Investigating the connection between complexity of isolated trajectories and Lagrangian coherent structures

    NASA Astrophysics Data System (ADS)

    Rypina, I. I.; Scott, S. E.; Pratt, L. J.; Brown, M. G.

    2011-12-01

    It is argued that the complexity of fluid particle trajectories provides the basis for a new method, referred to as the Complexity Method (CM), for estimation of Lagrangian coherent structures in aperiodic flows that are measured over finite time intervals. The basic principles of the CM are explained and the CM is tested in a variety of examples, both idealized and realistic, and in different reference frames. Two measures of complexity are explored in detail: the correlation dimension of trajectory, and a new measure - the ergodicity defect. Both measures yield structures that strongly resemble Lagrangian coherent structures in all of the examples considered. Since the CM uses properties of individual trajectories, and not separation rates between closely spaced trajectories, it may have advantages for the analysis of ocean float and drifter data sets in which trajectories are typically widely and non-uniformly spaced.

  1. Predicting Electrophoretic Mobility of Protein-Ligand Complexes for Ligands from DNA-Encoded Libraries of Small Molecules.

    PubMed

    Bao, Jiayin; Krylova, Svetlana M; Cherney, Leonid T; Hale, Robert L; Belyanskaya, Svetlana L; Chiu, Cynthia H; Shaginian, Alex; Arico-Muendel, Christopher C; Krylov, Sergey N

    2016-05-17

    Selection of target-binding ligands from DNA-encoded libraries of small molecules (DELSMs) is a rapidly developing approach in drug-lead discovery. Methods of kinetic capillary electrophoresis (KCE) may facilitate highly efficient homogeneous selection of ligands from DELSMs. However, KCE methods require accurate prediction of electrophoretic mobilities of protein-ligand complexes. Such prediction, in turn, requires a theory that would be applicable to DNA tags of different structures used in different DELSMs. Here we present such a theory. It utilizes a model of a globular protein connected, through a single point (small molecule), to a linear DNA tag containing a combination of alternating double-stranded and single-stranded DNA (dsDNA and ssDNA) regions of varying lengths. The theory links the unknown electrophoretic mobility of protein-DNA complex with experimentally determined electrophoretic mobilities of the protein and DNA. Mobility prediction was initially tested by using a protein interacting with 18 ligands of various combinations of dsDNA and ssDNA regions, which mimicked different DELSMs. For all studied ligands, deviation of the predicted mobility from the experimentally determined value was within 11%. Finally, the prediction was tested for two proteins and two ligands with a DNA tag identical to those of DELSM manufactured by GlaxoSmithKline. Deviation between the predicted and experimentally determined mobilities did not exceed 5%. These results confirm the accuracy and robustness of our model, which makes KCE methods one step closer to their practical use in selection of drug leads, and diagnostic probes from DELSMs. PMID:27119259

  2. 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.

  3. Novel Cul3 binding proteins function to remodel E3 ligase complexes

    PubMed Central

    2014-01-01

    Background Cullins belong to a family of scaffold proteins that assemble multi-subunit ubiquitin ligase complexes to recruit protein substrates for ubiquitination via unique sets of substrate adaptor, such as Skp1 or Elongin B, and a substrate-binding protein with a conserved protein-protein interacting domain, such as leucine-rich repeats (LRR), a WD40 domain, or a zinc-finger domain. In the case of the Cullin3 (Cul3), it forms a BTB-Cul3-Rbx1 (BCR) ubiquitin ligase complex where it is believed that a BTB domain-containing protein performs dual functions where it serves as both the substrate adaptor and the substrate recognition protein. Results Tandem affinity purification and LC/MS-MS analysis of the BCR complex led to the identification of 10,225 peptides. After the SEQUEST algorithm and CDART program were used for protein identification and domain prediction, we discovered a group of Cul3-bound proteins that contain either the LRR or WD40 domain (CLWs). Further biochemical analysis revealed that the LRR domain-containing CLWs could bind both Cul3 and BTB domain-containing proteins. The dual binding role for the LRR domain-containing CLWs results in causing the BTB-domain protein to become a substrate instead of an adaptor. To further distinguish potential substrates from other components that are part of the BCR ubiquitin ligase complex, we altered the parameters in the SEQUEST algorithm to select for peptide fragments with a modified lysine residue. This method not only identifies the potential substrates of the BCR ubiquitin ligase complex, but it also pinpoints the lysine residue in which the post-translational modification occurs. Interestingly, none of the CLWs were identified by this method, supporting our hypothesis that CLWs were not potential substrates but rather additional components of the BCR ubiquitin ligase complex. Conclusion Our study identified a new set of Cul3-binding proteins known as CLWs via tandem affinity purification and LC

  4. Restricted dynamics of water around a protein-carbohydrate complex: Computer simulation studies

    NASA Astrophysics Data System (ADS)

    Jana, Madhurima; Bandyopadhyay, Sanjoy

    2012-08-01

    Water-mediated protein-carbohydrate interaction is a complex phenomenon responsible for different biological processes in cellular environment. One of the unexplored but important issues in this area is the role played by water during the recognition process and also in controlling the microscopic properties of the complex. In this study, we have carried out atomistic molecular dynamics simulations of a protein-carbohydrate complex formed between the hyaluronan binding domain of the murine Cd44 protein and the octasaccharide hyaluronan in explicit water. Efforts have been made to explore the heterogeneous influence of the complex on the dynamic properties of water present in different regions around it. It is revealed from our analyses that the heterogeneous dynamics of water around the complex are coupled with differential time scales of formation and breaking of hydrogen bonds at the interface. Presence of a highly rigid thin layer of motionally restricted water molecules bridging the protein and the carbohydrate in the common region of the complex has been identified. Such water molecules are expected to play a crucial role in controlling properties of the complex. Importantly, it is demonstrated that the formation of the protein-carbohydrate complex affects the transverse and longitudinal degrees of freedom of the interfacial water molecules in a heterogeneous manner.

  5. Signal peptide peptidase (SPP) assembles with substrates and misfolded membrane proteins into distinct oligomeric complexes

    PubMed Central

    Schrul, Bianca; Kapp, Katja; Sinning, Irmgard; Dobberstein, Bernhard

    2010-01-01

    SPP (signal peptide peptidase) is an aspartyl intramembrane cleaving protease, which processes a subset of signal peptides, and is linked to the quality control of ER (endoplasmic reticulum) membrane proteins. We analysed SPP interactions with signal peptides and other membrane proteins by co-immunoprecipitation assays. We found that SPP interacts specifically and tightly with a large range of newly synthesized membrane proteins, including signal peptides, preproteins and misfolded membrane proteins, but not with all co-expressed type II membrane proteins. Signal peptides are trapped by the catalytically inactive SPP mutant SPPD/A. Preproteins and misfolded membrane proteins interact with both SPP and the SPPD/A mutant, and are not substrates for SPP-mediated intramembrane proteolysis. Proteins interacting with SPP are found in distinct complexes of different sizes. A signal peptide is mainly trapped in a 200 kDa SPP complex, whereas a preprotein is predominantly found in a 600 kDa SPP complex. A misfolded membrane protein is detected in 200, 400 and 600 kDa SPP complexes. We conclude that SPP not only processes signal peptides, but also collects preproteins and misfolded membrane proteins that are destined for disposal. PMID:20196774

  6. 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.

  7. 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…

  8. 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…

  9. Quantification of dynamic protein complexes using Renilla luciferase fragment complementation applied to protein kinase A activities in vivo

    PubMed Central

    Stefan, E.; Aquin, S.; Berger, N.; Landry, C. R.; Nyfeler, B.; Bouvier, M.; Michnick, S. W.

    2007-01-01

    The G protein-coupled receptor (GPCR) superfamily represents the most important class of pharmaceutical targets. Therefore, the characterization of receptor cascades and their ligands is a prerequisite to discovering novel drugs. Quantification of agonist-induced second messengers and downstream-coupled kinase activities is central to characterization of GPCRs or other pathways that converge on GPCR-mediated signaling. Furthermore, there is a need for simple, cell-based assays that would report on direct or indirect actions on GPCR-mediated effectors of signaling. More generally, there is a demand for sensitive assays to quantify alterations of protein complexes in vivo. We describe the development of a Renilla luciferase (Rluc)-based protein fragment complementation assay (PCA) that was designed specifically to investigate dynamic protein complexes. We demonstrate these features for GPCR-induced disassembly of protein kinase A (PKA) regulatory and catalytic subunits, a key effector of GPCR signaling. Taken together, our observations show that the PCA allows for direct and accurate measurements of live changes of absolute values of protein complex assembly and disassembly as well as cellular imaging and dynamic localization of protein complexes. Moreover, the Rluc-PCA has a sufficiently high signal-to-background ratio to identify endogenously expressed Gαs protein-coupled receptors. We provide pharmacological evidence that the phosphodiesterase-4 family selectively down-regulates constitutive β-2 adrenergic- but not vasopressin-2 receptor-mediated PKA activities. Our results show that the sensitivity of the Rluc-PCA simplifies the recording of pharmacological profiles of GPCR-based candidate drugs and could be extended to high-throughput screens to identify novel direct modulators of PKA or upstream components of GPCR signaling cascades. PMID:17942691

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

    PubMed Central

    Rappsilber, Juri

    2011-01-01

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

  11. Fractal and complex network analyses of protein molecular dynamics

    NASA Astrophysics Data System (ADS)

    Zhou, Yuan-Wu; Liu, Jin-Long; Yu, Zu-Guo; Zhao, Zhi-Qin; Anh, Vo

    2014-12-01

    Based on protein molecular dynamics, we investigate the fractal properties of energy, pressure and volume time series using the multifractal detrended fluctuation analysis (MF-DFA) and the topological and fractal properties of their converted horizontal visibility graphs (HVGs). The energy parameters of protein dynamics we considered are bonded potential, angle potential, dihedral potential, improper potential, kinetic energy, Van der Waals potential, electrostatic potential, total energy and potential energy. The shape of the h(q) curves from MF-DFA indicates that these time series are multifractal. The numerical values of the exponent h(2) of MF-DFA show that the series of total energy and potential energy are non-stationary and anti-persistent; the other time series are stationary and persistent apart from series of pressure (with H≈0.5 indicating the absence of long-range correlation). The degree distributions of their converted HVGs show that these networks are exponential. The results of fractal analysis show that fractality exists in these converted HVGs. For each energy, pressure or volume parameter, it is found that the values of h(2) of MF-DFA on the time series, exponent λ of the exponential degree distribution and fractal dimension dB of their converted HVGs do not change much for different proteins (indicating some universality). We also found that after taking average over all proteins, there is a linear relationship between (from MF-DFA on time series) and of the converted HVGs for different energy, pressure and volume.

  12. The Usher syndrome proteins cadherin 23 and harmonin form a complex by means of PDZ-domain interactions

    PubMed Central

    Siemens, Jan; Kazmierczak, Piotr; Reynolds, Anna; Sticker, Melanie; Littlewood-Evans, Amanda; Müller, Ulrich

    2002-01-01

    Usher syndrome type 1 (USH1) patients suffer from sensorineuronal deafness, vestibular dysfunction, and visual impairment. Several genetic loci have been linked to USH1, and four of the relevant genes have been identified. They encode the unconventional myosin VIIa, the PDZ-domain protein harmonin, and the putative adhesion receptors cadherin 23 (CDH23) and protocadherin 15 (PCDH15). We show here that CDH23 and harmonin form a protein complex. Two PDZ domains in harmonin interact with two complementary binding surfaces in the CDH23 cytoplasmic domain. One of the binding surfaces is disrupted by sequences encoded by an alternatively spliced CDH23 exon that is expressed in the ear, but not the retina. In the ear, CDH23 and harmonin are expressed in the stereocilia of hair cells, and in the retina within the photoreceptor cell layer. Because CDH23-deficient mice have splayed stereocilia, our data suggest that CDH23 and harmonin are part of a transmembrane complex that connects stereocilia into a bundle. Defects in the formation of this complex are predicted to disrupt stereocilia bundles and cause deafness in USH1 patients. PMID:12407180

  13. 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

  14. Capillary electrophoresis methods for the determination of covalent polyphenol-protein complexes.

    PubMed

    Trombley, John D; Loegel, Thomas N; Danielson, Neil D; Hagerman, Ann E

    2011-09-01

    The bioactivities and bioavailability of plant polyphenols including proanthocyanidins and other catechin derivatives may be affected by covalent reaction between polyphenol and proteins. Both processing conditions and gastrointestinal conditions may promote formation of covalent complexes for polyphenol-rich foods and beverages such as wine. Little is known about covalent reactions between proteins and tannin, because suitable methods for quantitating covalent complexes have not been developed. We established capillary electrophoresis methods that can be used to distinguish free protein from covalently bound protein-polyphenol complexes and to monitor polyphenol oxidation products. The methods are developed using the model protein bovine serum albumin and the representative polyphenol (-)epigallocatechin gallate. By pairing capillaries with different diameters with appropriate alkaline borate buffers, we are able to optimize resolution of either the protein-polyphenol complexes or the polyphenol oxidation products. This analytical method, coupled with purification of the covalent complexes by diethylaminoethyl cellulose chromatography, should facilitate characterization of covalent complexes in polyphenol-rich foods and beverages such as wine. PMID:21400190

  15. An integrated approach to identify protein complex based on best neighbour and modularity increment.

    PubMed

    Shen, Xianjun; Zhao, Yanli; Li, Yanan; Yi, Yang; He, Tingting; Yang, Jincai

    2015-01-01

    In order to overcome the limitations of global modularity and the deficiency of local modularity, we propose a hybrid modularity measure Local-Global Quantification (LGQ) which considers global modularity and local modularity together. LGQ adopts a suitable module feature adjustable parameter to control the balance of global detecting capability and local search capability in Protein-Protein Interactions (PPI) Network. Furthermore, we develop a new protein complex mining algorithm called Best Neighbour and Local-Global Quantification (BN-LGQ) which integrates the best neighbour node and modularity increment. BN-LGQ expands the protein complex by fast searching the best neighbour node of the current cluster and by calculating the modularity increment as a metric to determine whether the best neighbour node can join the current cluster. The experimental results show BN-LGQ performs a better accuracy on predicting protein complexes and has a higher match with the reference protein complexes than MCL and MCODE algorithms. Moreover, BN-LGQ can effectively discover protein complexes with better biological significance in the PPI network. PMID:26336669

  16. 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. PMID:25803397

  17. 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

  18. Wrap-and-Strip Technology of Protein-Polyelectrolyte Complex for Biomedical Application.

    PubMed

    Shiraki, Kentaro; Kurinomaru, Takaaki; Tomita, Shunsuke

    2016-01-01

    A polyelectrolyte is a polymer composed of repeating units of an electrolyte group that enables reversible complex formation with proteins in aqueous solutions. This review introduces "wrap-and-strip" technology of protein-polyelectrolyte complex (PPC) by noncovalent interaction. Storage: protein is stabilized against physical and chemical stresses. Enrichment: precipitation through PPC can be used as an enrichment method without irreversible unfolding. Catalytic activity switch: a complementary charged pair of polyelectrolytes functions as a reversible enzyme activity switch. Hyperactivation: a specific combination of a polyelectrolyte and substrate enhances enzyme activity by one order of magnitude compared with an enzyme alone. Stabilization: PPC increases protein stability against chemical and physical stresses, such as covalently modified polyethylene glycosylated protein. Simple PPC-based technology can expand the applicable fields of soluble proteins in aqueous solutions. PMID:26630921

  19. 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

  20. A Complex of Bir1-Sli15 (Survivin-INCENP) Connects Centromeres to Microtubules and is the Likely Tension Sensor Controlling Aurora B Activation

    PubMed Central

    Sandall, Sharsti; Severin, Fedor; McLeod, Ian X.; Yates, John R.; Oegema, Karen; Hyman, Anthony; Desai1, Arshad

    2007-01-01

    SUMMARY Proper connections between centromeres and spindle microtubules are of critical importance in ensuring accurate segregation of the genome during cell division. Using an in vitro approach based on the sequence-specific budding yeast centromere, we identified a complex of the chromosomal passenger proteins Bir1 and Sli15 (Survivin and INCENP) that links centromeres to microtubules. This linkage does not require Ipl1/Aurora B kinase, whose targeting and activation are controlled by Bir1 and Sli15. Ipl1 is the tension-dependent regulator of centromere-microtubule interactions that ensures chromosome bi-orientation on the spindle. Elimination of the linkage between centromeres and microtubules mediated by Bir1-Sli15 phenocopies mutations that selectively cripple Ipl1 kinase activation. These findings lead us to propose that the Bir1-Sli15 mediated linkage, which bridges centromeres and microtubules and includes the Aurora kinase-activating domain of INCENP family proteins, is the tension sensor that relays the mechanical state of centromere-microtubule attachments into local control of Ipl1 kinase activity. PMID:17174893

  1. TssK Is a Trimeric Cytoplasmic Protein Interacting with Components of Both Phage-like and Membrane Anchoring Complexes of the Type VI Secretion System*

    PubMed Central

    Zoued, Abdelrahim; Durand, Eric; Bebeacua, Cecilia; Brunet, Yannick R.; Douzi, Badreddine; Cambillau, Christian; Cascales, Eric; Journet, Laure

    2013-01-01

    The Type VI secretion system (T6SS) is a macromolecular machine that mediates bacteria-host or bacteria-bacteria interactions. The T6SS core apparatus assembles from 13 proteins that form two sub-assemblies: a phage-like complex and a trans-envelope complex. The Hcp, VgrG, TssE, and TssB/C subunits are structurally and functionally related to components of the tail of contractile bacteriophages. This phage-like structure is thought to be anchored to the membrane by a trans-envelope complex composed of the TssJ, TssL, and TssM proteins. However, how the two sub-complexes are connected remains unknown. Here we identify TssK, a protein that establishes contacts with the two T6SS sub-complexes through direct interactions with TssL, Hcp, and TssC. TssK is a cytoplasmic protein assembling trimers that display a three-armed shape, as revealed by TEM and SAXS analyses. Fluorescence microscopy experiments further demonstrate the requirement of TssK for sheath assembly. Our results suggest a central role for TssK by linking both complexes during T6SS assembly. PMID:23921384

  2. 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

  3. Robust assessment of protein complex formation in vivo via single-molecule intensity distributions of autofluorescent proteins

    NASA Astrophysics Data System (ADS)

    Meckel, Tobias; Semrau, Stefan; Schaaf, Marcel J. M.; Schmidt, Thomas

    2011-07-01

    The formation of protein complexes or clusters in the plasma membrane is essential for many biological processes, such as signaling. We develop a tool, based on single-molecule microscopy, for following cluster formation in vivo. Detection and tracing of single autofluorescent proteins have become standard biophysical techniques. The determination of the number of proteins in a cluster, however, remains challenging. The reasons are (i) the poor photophysical stability and complex photophysics of fluorescent proteins and (ii) noise and autofluorescent background in live cell recordings. We show that, despite those obstacles, the accurate fraction of signals in which a certain (or set) number of labeled proteins reside, can be determined in an accurate an robust way in vivo. We define experimental conditions under which fluorescent proteins exhibit predictable distributions of intensity and quantify the influence of noise. Finally, we confirm our theoretical predictions by measurements of the intensities of individual enhanced yellow fluorescent protein (EYFP) molecules in living cells. Quantification of the average number of EYFP-C10HRAS chimeras in diffraction-limited spots finally confirm that the membrane anchor of human Harvey rat sarcoma (HRAS) heterogeneously distributes in the plasma membrane of living Chinese hamster ovary cells.

  4. 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

  5. An informatic framework for decoding protein complexes by top-down mass spectrometry.

    PubMed

    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

    2016-03-01

    Efforts to map the human protein interactome have resulted in information about thousands of multi-protein assemblies housed in public repositories, but the molecular characterization and stoichiometry of their protein subunits remains largely unknown. Here, we report a computational search strategy that supports hierarchical top-down analysis for precise identification and scoring of multi-proteoform complexes by native mass spectrometry. PMID:26780093

  6. Quantitative assessment of complex formation of nuclear-receptor accessory proteins.

    PubMed

    Graumann, K; Jungbauer, A

    2000-02-01

    Like other nuclear receptors, steroid hormone receptors form large protein hetero-complexes in their inactive, ligand-friendly state. Several heat-shock proteins, immunophilins and others have been identified as members of these highly dynamic complexes. The interaction kinetics and dynamics of hsp90, hsp70, p60 (Hop), FKBP52, FKBP51, p48 (Hip) and p23 have been assessed by a biosensor approach measuring the complex formation in real time. A core chaperone complex has been reconstituted from p60, hsp90 and hsp70. p60 forms a molecular bridge between hsp90 and hsp70 with an affinity in the range of 10(5) M(-1). Dynamics of hsp90-p60 complex formation is modulated by ATP through changes in the co-operativity of interaction. At low protein concentrations ATP stabilizes the complex. Binding of p23 to hsp90 did not change the affinity of the hsp90-p60 complex and the stabilizing effect of ATP. Saturation of the p48-hsp70 interaction could not be achieved, suggesting multiple binding sites. A picture of the protein complex, including stoichiometric coefficients, co-operativity of interaction and equilibrium-binding constants, has been formed. PMID:10642522

  7. Human-chromatin-related protein interactions identify a demethylase complex required for chromosome segregation.

    PubMed

    Marcon, Edyta; Ni, Zuyao; Pu, Shuye; Turinsky, Andrei L; Trimble, Sandra Smiley; Olsen, Jonathan B; Silverman-Gavrila, Rosalind; Silverman-Gavrila, Lorelei; Phanse, Sadhna; Guo, Hongbo; Zhong, Guoqing; Guo, Xinghua; Young, Peter; Bailey, Swneke; Roudeva, Denitza; Zhao, Dorothy; Hewel, Johannes; Li, Joyce; Gräslund, Susanne; Paduch, Marcin; Kossiakoff, Anthony A; Lupien, Mathieu; Emili, Andrew; Wodak, Shoshana J; Greenblatt, Jack

    2014-07-10

    Chromatin regulation is driven by multicomponent protein complexes, which form functional modules. Deciphering the components of these modules and their interactions is central to understanding the molecular pathways these proteins are regulating, their functions, and their relation to both normal development and disease. We describe the use of affinity purifications of tagged human proteins coupled with mass spectrometry to generate a protein-protein interaction map encompassing known and predicted chromatin-related proteins. On the basis of 1,394 successful purifications of 293 proteins, we report a high-confidence (85% precision) network involving 11,464 protein-protein interactions among 1,738 different human proteins, grouped into 164 often overlapping protein complexes with a particular focus on the family of JmjC-containing lysine demethylases, their partners, and their roles in chromatin remodeling. We show that RCCD1 is a partner of histone H3K36 demethylase KDM8 and demonstrate that both are important for cell-cycle-regulated transcriptional repression in centromeric regions and accurate mitotic division. PMID:24981860

  8. The biological activities of protein/oleic acid complexes reside in the fatty acid.

    PubMed

    Fontana, Angelo; Spolaore, Barbara; Polverino de Laureto, Patrizia

    2013-06-01

    A complex formed by human α-lactalbumin (α-LA) and oleic acid (OA), named HAMLET, has been shown to have an apoptotic activity leading to the selective death of tumor cells. In numerous publications it has been reported that in the complex α-LA is monomeric and adopts a partly folded or "molten globule" state, leading to the idea that partly folded proteins can have "beneficial effects". The protein/OA molar ratio initially has been reported to be 1:1, while recent data have indicated that the OA-complex is given by an oligomeric protein capable of binding numerous OA molecules per protein monomer. Proteolytic fragments of α-LA, as well as other proteins unrelated to α-LA, can form OA-complexes with biological activities similar to those of HAMLET, thus indicating that a generic protein can form a cytotoxic complex under suitable experimental conditions. Moreover, even the selective tumoricidal activity of HAMLET-like complexes has been questioned. There is recent evidence that the biological activity of long chain unsaturated fatty acids, including OA, can be ascribed to their effect of perturbing the structure of biological membranes and consequently the function of membrane-bound proteins. In general, it has been observed that the cytotoxic effects exerted by HAMLET-like complexes are similar to those reported for OA alone. Overall, these findings can be interpreted by considering that the protein moiety does not have a toxic effect on its own, but merely acts as a solubilising agent for the inherently toxic fatty acid. PMID:23499846

  9. Unraveling the Complexities of DNA-Dependent Protein Kinase Autophosphorylation

    PubMed Central

    Neal, Jessica A.; Sugiman-Marangos, Seiji; VanderVere-Carozza, Pamela; Wagner, Mike; Turchi, John; Lees-Miller, Susan P.; Junop, Murray S.

    2014-01-01

    DNA-dependent protein kinase (DNA-PK) orchestrates DNA repair by regulating access to breaks through autophosphorylations within two clusters of sites (ABCDE and PQR). Blocking ABCDE phosphorylation (by alanine mutation) imparts a dominant negative effect, rendering cells hypersensitive to agents that cause DNA double-strand breaks. Here, a mutational approach is used to address the mechanistic basis of this dominant negative effect. Blocking ABCDE phosphorylation hypersensitizes cells to most types of DNA damage (base damage, cross-links, breaks, and damage induced by replication stress), suggesting that DNA-PK binds DNA ends that result from many DNA lesions and that blocking ABCDE phosphorylation sequesters these DNA ends from other repair pathways. This dominant negative effect requires DNA-PK's catalytic activity, as well as phosphorylation of multiple (non-ABCDE) DNA-PK catalytic subunit (DNA-PKcs) sites. PSIPRED analysis indicates that the ABCDE sites are located in the only contiguous extended region of this huge protein that is predicted to be disordered, suggesting a regulatory role(s) and perhaps explaining the large impact ABCDE phosphorylation has on the enzyme's function. Moreover, additional sites in this disordered region contribute to the ABCDE cluster. These data, coupled with recent structural data, suggest a model whereby early phosphorylations promote initiation of nonhomologous end joining (NHEJ), whereas ABCDE phosphorylations, potentially located in a “hinge” region between the two domains, lead to regulated conformational changes that initially promote NHEJ and eventually disengage NHEJ. PMID:24687855

  10. Viscoelastic properties of electrochemically deposited protein/metal complexes.

    PubMed

    Martin, Elizabeth J; Mathew, Mathew T; Shull, Kenneth R

    2015-04-01

    The interfacial gelation of proteins at metallic surfaces was investigated with an electrochemical quartz crystal microbalance (QCM). When Cr electrodes were corroded in proteinaceous solutions, it was found that gels will form at the Cr surfaces if molybdate ions are also present in the solution. Gelation is reversible and can also be controlled with the electrochemical potential at the electrode. Further, a method was developed to characterize the viscoelastic properties of thin films in liquid media using the QCM as a high-frequency rheometer. By measuring the frequency and dissipation at multiple harmonics of the resonance frequency, the viscoelastic phase angle, density-modulus product, and areal mass of a film can be determined. The method was applied to characterize the protein films, demonstrating that they have a phase angle near 55° and a density-modulus product of ≈10(7) Pa·g/cm(3). Data imply that the gels are composed of a weakly cross-linked proteinaceous network with properties similar to albumin solutions with concentrations in the range of ≈40 wt %. PMID:25780816

  11. Forisomes as calcium-energized protein complex: a historical perspective.

    PubMed

    Tuteja, Narendra; Umate, Pavan; Tuteja, Renu

    2010-05-01

    Forisomes are spindle-shaped (about 1-3 µm wide and 10-30 µm long) contractile moving protein bodies found exclusively in phloem sieve tubes of Fabaceae (legume) plants. They are composed of ATP-independent, mechanically active proteins with several subunits called "forisomettes" and play important role in plant defence against injury. They expand and contract reversibly independent of ATP in response to changes of concentration of Ca2+ ions and pH or electric field. Although forisomes have been discovered decades ago, but only recently they received broader attention due to their ability to convert chemical into mechanical energy. Forisomes have possible applications as biomimetic smart materials (e.g. valves in microdevices), or smart composite materials for nanotechnological applications. Forisome-based smart materials can be used to synthesize self-powered monitoring and diagnostic systems for health monitoring. Here we discuss precise overview of forisomes from a historical viewpoint. We also enlist the scientific events leading to the identification and characterization of forisomes and their potential applications. The advancements in forisomes research have marked the evolution of new trends in the field of applied 'biomimetics' (means: to 'mimic life'). PMID:20215879

  12. Protein complex analysis of native brain potassium channels by proteomics.

    PubMed

    Sandoz, Guillaume; Lesage, Florian

    2008-01-01

    TREK potassium channels belong to a family of channel subunits with two-pore domains (K(2P)). TREK1 knockout mice display impaired polyunsaturated fatty acid-mediated protection against brain ischemia, reduced sensitivity to volatile anesthetics, resistance to depression and altered perception of pain. Recently, we isolated native TREK1 channels from mouse brain and identified their specific components by mass spectrometry. Among the identified partners, the A-Kinase Anchoring Protein AKAP150 binds to a regulatory domain of TREK1 and acts as a molecular switch. It transforms low activity, outwardly rectifying TREK1 currents into robust leak conductances resistant to stimulation by arachidonic acid, membrane stretch and acidification. Inhibition of the TREK1/AKAP150 channel by Gs-coupled receptors is as extensive as for TREK1 alone (but faster) whereas inhibition of TREK1/AKAP150 by Gq-coupled receptors is reduced. Furthermore, the association of AKAP150 with TREK1 channels integrates them into postsynaptic scaffolds where G protein-coupled membrane receptors and channels dock simultaneously. This chapter describes the proteomic approach used to study the composition of native TREK1 channels and point out its advantages and limitations over more classical methods (two-hybrid screenings in the yeast and bacteria or GST-pull down). PMID:18998088

  13. Lateral heterogeneity of plant thylakoid protein complexes: early reminiscences

    PubMed Central

    Anderson, Jan M.

    2012-01-01

    The concept that the two photosystems of photosynthesis cooperate in series, immortalized in Hill and Bendall's Z scheme, was still a black box that defined neither the structural nor the molecular organization of the thylakoid membrane network into grana and stroma thylakoids. The differentiation of the continuous thylakoid membrane into stacked grana thylakoids interconnected by single stroma thylakoids is a morphological reflection of the non-random distribution of photosystem II/light-harvesting complex of photosystem II, photosystem I and ATP synthase, which became known as lateral heterogeneity. PMID:23148264

  14. 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

  15. Supporting decision-making for improving longitudinal connectivity for diadromous and potamodromous fishes in complex catchments.

    PubMed

    Brevé, Niels W P; Buijse, Anthonie D; Kroes, Martin J; Wanningen, Herman; Vriese, Frederik T

    2014-10-15

    Preservation and restoration of Europe's endangered migratory fish species and habitats are high on the international river basin policy agenda. Improvement through restoration of longitudinal connectivity is seen as an important measure, but although prioritization of in-stream barriers has been addressed at local and regional levels the process still lacks adequate priority on the international level. This paper introduces a well-tested method, designed to help decision makers achieve the rehabilitation of targeted ichthyofauna more successfully. This method assesses artificial barriers within waters designated under the Water Framework Directive (WFD), Europe's main legislative driver for ecological improvement of river basins. The method aggregates migratory fish communities (both diadromous and potamodromous) into functional biological units (ecological fish guilds) and defines their most pressing habitat requirements. Using GIS mapping and spatial analysis of the potential ranges (fish zonation) we pin-point the most important barriers, per guild. This method was developed and deployed over a 12 year period as a practical case study, fitting data derived from the 36 regional water management organisations in the Netherlands. We delivered national advice on the prioritization of a total of 2924 barriers located within WFD water bodies, facilitating migration for all 18 indigenous migratory fish species. Scaling up to larger geographical areas can be achieved using datasets from other countries to link water body typologies to distribution ranges of migratory fish species. PMID:25084228

  16. 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

  17. Work complexity assessment, nursing interventions classification, and nursing outcomes classification: making connections.

    PubMed

    Scherb, Cindy A; Weydt, Alice P

    2009-01-01

    When nurses understand what interventions are needed to achieve desired patient outcomes, they can more easily define their practice. Work Complexity Assessment (WCA) is a process that helps nurses to identify interventions performed on a routine basis for their specific patient population. This article describes the WCA process and links it to the Nursing Interventions Classification (NIC) and the Nursing Outcomes Classification (NOC). WCA, NIC, and NOC are all tools that help nurses understand the work they do and the outcomes they achieve, and that thereby acknowledge and validate nursing's contribution to patient care. PMID:19343845

  18. 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.

  19. 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

  20. 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

  1. 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.

  2. 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.

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

    PubMed

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

    2005-08-01

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

  4. Advanced biomolecular materials based on membrane-protein/polymer complexation

    SciTech Connect

    Smith, G.S.; Nowak, A.; Safinya, C.

    1998-12-01

    This is the final report of a three-year, Laboratory Directed Research and Development (LDRD) project at the Los Alamos National Laboratory (LANL). The goal of this project was to apply neutron reflectometry and atomic force microscopy to the study of lipid membranes containing proteins. Standard sample preparation techniques were used to produce thin films of these materials appropriate for these techniques. However, these films were not stable, and a new sample preparation technique was required. Toward this goal, the authors have developed a new capability to produce large, freely suspended films of lipid multi-bilayers appropriate for these studies. This system includes a controlled temperature/humidity oven in which the films 5-cm x 5-cm are remotely drawn. The first neutron scattering experiments were then performed using this oven.

  5. Gold Nanoparticle-Polymer/Biopolymer Complexes for Protein Sensing

    PubMed Central

    Moyano, Daniel F.; Rana, Subinoy; Bunz, Uwe H. F.; Rotello, Vincent M.

    2014-01-01

    Nanoparticle-based sensor arrays have been used to distinguish a wide range of biomolecular targets through pattern recognition. Such biosensors require selective receptors that generate a unique response pattern for each analyte. The tunable surface properties of gold nanoparticles make these systems excellent candidates for the recognition process. Likewise, the metallic core makes these particles fluorescence superquenchers, facilitating transduction of the binding event. In this report we analyze the role of gold nanoparticles as receptors in differentiating a diversity of important human proteins different, and the role of the polymer/biopolymer fluorescent probes for transducing the binding event. A structure-activity relationship analysis of both the probes and the nanoparticles is presented, providing direction for the engineering of future sensor systems. PMID:22455037

  6. 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 Central

    Masuho, Ikuo; Xie, Keqiang; Martemyanov, Kirill A.

    2013-01-01

    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. PMID:23857581

  7. The Fine Structure of Experimentally Induced Connective Tissue Complexes in the Human

    PubMed Central

    Stinson, W. W.; Richter, K. M.; Schilling, J. A.

    1974-01-01

    The growth, development and cellular activity of fibrocollagenous tissue complexes induced by the implantation of specially structured wire mesh cylinders in 22 human male volunteers were studied during the time course of fibroplasia utilizing light and electron microscopic techniques. The fibrocollagenous tissue complexes after 4 to 16 weeks of development demonstrated highly ordered lamellations made of zones consisting primarily of fibroblasts and zones consisting primarily of collagenous fibers. The development of the ordered lamellations is referable to specific fibrillogenic activities by the constituent fibroblasts. The initial role of the fibroblast in fibrillogenesis is indicative of an apocrine-like secretory process followed by a holocrine-like role which results in cytodestruction and concurrent formation of an avascular collagenic tissue referable to an organizing cicatrix in a healing wound. ImagesFigs. 1-3.Figs. 4-9.Fig. 10.Fig. 11.Fig. 12.Figs. 13, 14.Fig. 15.Fig. 16.Fig. 17.Fig. 18, 19.Figs. 20-22.Fig. 23.Fig. 24. PMID:4599224

  8. Global and local structural similarity in protein–protein complexes: Implications for template-based docking

    PubMed Central

    Kundrotas, Petras J.; Vakser, Ilya A.

    2016-01-01

    The increasing amount of structural information on protein–protein interactions makes it possible to predict the structure of protein–protein complexes by comparison/alignment of the interacting proteins to the ones in cocrystallized complexes. In the predictions based on structure similarity, the template search is performed by structural alignment of the target interactors with the entire structures or with the interface only of the subunits in cocrystallized complexes. This study investigates the scope of the structural similarity that facilitates the detection of a broad range of templates significantly divergent from the targets. The analysis of the target-template similarity is based on models of protein–protein complexes in a large representative set of heterodimers. The similarity of the biological and crystal packing interfaces, dissimilar interface structural motifs in overall similar structures, interface similarity to the full structure, and local similarity away from the interface were analyzed. The structural similarity at the protein–protein interfaces only was observed in ~25% of target-template pairs with sequence identity <20% and primarily homodimeric templates. For ~50% of the target-template pairs, the similarity at the interface was accompanied by the similarity of the whole structure. However, the structural similarity at the interfaces was still stronger than that of the noninterface parts. The study provides insights into structural and functional diversity of protein–protein complexes, and relative performance of the interface and full structure alignment in docking. PMID:23946125

  9. Sec1/Munc18 protein Vps33 binds to SNARE domains and the quaternary SNARE complex

    PubMed Central

    Lobingier, Braden T.; Merz, Alexey J.

    2012-01-01

    Soluble N-ethylmaleimide–sensitive factor attachment protein receptor (SNARE) proteins catalyze membrane fusion events in the secretory and endolysosomal systems, and all SNARE-mediated fusion processes require cofactors of the Sec1/Munc18 (SM) family. Vps33 is an SM protein and subunit of the Vps-C complexes HOPS (homotypic fusion and protein sorting) and CORVET (class C core vacuole/endosome tethering), which are central regulators of endocytic traffic. Here we present biochemical studies of interactions between Saccharomyces cerevisiae vacuolar SNAREs and the HOPS holocomplex or Vps33 alone. HOPS binds the N-terminal Habc domain of the Qa-family SNARE Vam3, but Vps33 is not required for this interaction. Instead, Vps33 binds the SNARE domains of Vam3, Vam7, and Nyv1. Vps33 directly binds vacuolar quaternary SNARE complexes, and the affinity of Vps33 for SNARE complexes is greater than for individual SNAREs. Through targeted mutational analyses, we identify missense mutations of Vps33 that produce a novel set of defects, including cargo missorting and the loss of Vps33-HOPS association. Together these data suggest a working model for membrane docking: HOPS associates with N-terminal domains of Vam3 and Vam7 through Vps33-independent interactions, which are followed by binding of Vps33, the HOPS SM protein, to SNARE domains and finally to the quaternary SNARE complex. Our results also strengthen the hypothesis that SNARE complex binding is a core attribute of SM protein function. PMID:23051737

  10. Role of the conserved oligomeric Golgi (COG) complex in protein glycosylation☆

    PubMed Central

    Smith, Richard D.; Lupashin, Vladimir V.

    2009-01-01

    The Golgi apparatus is a central hub for both protein and lipid trafficking/sorting and is also a major site for glycosylation in the cell. This organelle employs a cohort of peripheral membrane proteins and protein complexes to keep its structural and functional organization. The conserved oligomeric Golgi (COG) complex is an evolutionary conserved peripheral membrane protein complex that is proposed to act as a retrograde vesicle tethering factor in intra-Golgi trafficking. The COG protein complex consists of eight subunits, distributed in two lobes, Lobe A (Cog1–4) and Lobe B (Cog5–8). Malfunctions in the COG complex have a significant impact on processes such as protein sorting, glycosylation, and Golgi integrity. A deletion of Lobe A COG subunits in yeasts causes severe growth defects while mutations in COG1, COG7, and COG8 in humans cause novel types of congenital disorders of glycosylation. These pathologies involve a change in structural Golgi phenotype and function. Recent results indicate that down-regulation of COG function results in the resident Golgi glycosyltransferases/glycosidases to be mislocalized or degraded. PMID:18353293

  11. Reactivity and applications of new amine reactive cross-linkers for mass spectrometric detection of protein-protein complexes.

    PubMed

    Bich, Claudia; Maedler, Stefanie; Chiesa, Katja; DeGiacomo, Fabio; Bogliotti, Nicolas; Zenobi, Renato

    2010-01-01

    Chemical cross-linking of proteins permits the stabilization of noncovalent complexes through introduction of covalent bonds. A crucial challenge is to find the fastest and most efficient cross-linkers in order to minimize reaction times and to handle delicate complexes. New cross-linkers were synthesized by introducing N-hydroxyphthalimide, hydroxybenzotriazole, and 1-hydroxy-7-azabenzotriazole as leaving groups instead of the commonly used N-hydroxysuccimidyl moiety. With the use of matrix-assisted laser desorption ionization (MALDI) mass spectrometry, these new cross-linkers were then compared with the commercially available disuccinimidyl suberate (DSS) for covalent stabilization of the gluthatione-S-transferase (GST) dimer and of an antibody-antigen complex. They showed a better efficiency, generated about 30% more cross-linked complex, and reacted about 10 times faster than DSS. The reaction with the GST dimer was utilized to get information about their reaction efficiency and kinetics. Their ability to stabilize only specific protein complexes was verified by incubating them with a mixture of the proteins GST and ubiquitin. Finally, the cross-linkers were incubated with synthetic peptides to study the selectivity of the binding with various amino acid side chains. Not only lysine but also tyrosine was found to react with the newly synthesized cross-linker containing 1-hydroxy-7-azabenzotriazole as the reactive group. PMID:19994840

  12. Structural consequences of effector protein complex formation in a diiron hydroxylase

    SciTech Connect

    Bailey, Lucas J.; McCoy, Jason G.; Phillips, Jr., George N.; Fox, Brian G.

    2009-06-12

    Carboxylate-bridged diiron hydroxylases are multicomponent enzyme complexes responsible for the catabolism of a wide range of hydrocarbons and as such have drawn attention for their mechanism of action and potential uses in bioremediation and enzymatic synthesis. These enzyme complexes use a small molecular weight effector protein to modulate the function of the hydroxylase. However, the origin of these functional changes is poorly understood. Here, we report the structures of the biologically relevant effector protein-hydroxylase complex of toluene 4-monooxygenase in 2 redox states. The structures reveal a number of coordinated changes that occur up to 25 {angstrom} from the active site and poise the diiron center for catalysis. The results provide a structural basis for the changes observed in a number of the measurable properties associated with effector protein binding. This description provides insight into the functional role of effector protein binding in all carboxylate-bridged diiron hydroxylases.

  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. Protein Labelling with Versatile Phosphorescent Metal Complexes for Live Cell Luminescence Imaging.

    PubMed

    Connell, Timothy U; James, Janine L; White, Anthony R; Donnelly, Paul S

    2015-09-28

    To take advantage of the luminescent properties of d(6) transition metal complexes to label proteins, versatile bifunctional ligands were prepared. Ligands that contain a 1,2,3-triazole heterocycle were synthesised using Cu(I) catalysed azide-alkyne cycloaddition "click" chemistry and were used to form phosphorescent Ir(III) and Ru(II) complexes. Their emission properties were readily tuned, by changing either the metal ion or the co-ligands. The complexes were tethered to the metalloprotein transferrin using several conjugation strategies. The Ir(III)/Ru(II)-protein conjugates could be visualised in cancer cells using live cell imaging for extended periods without significant photobleaching. These versatile phosphorescent protein-labelling agents could be widely applied to other proteins and biomolecules and are useful alternatives to conventional organic fluorophores for several applications. PMID:26264214

  15. Polypeptide composition of the purified photosystem II pigment-protein complex from spinach.

    PubMed

    Satoh, K

    1979-04-11

    The Photosystem II pigment-protein complex, the chlorophyll alpha-protein comprising the reaction center of Photosystem II, was prepared from EDTA-treated spinach chloroplasts by digitonin extraction, sucrose-gradient centrifugation, DEAE-cellulose column chromatography, and isoelectrofocussing on Ampholine. The dissociated pigment-protein complex exhibits two polypeptide subunits that migrate in SDS-polyacrylamide gel with electrophoretic mobilities corresponding to molecular weights of approximately 43,000 and 27,000. the chlorophyll was always found in the free pigment zone at the completion of the electrophoresis. Heat-treatment of the sample (100 degrees C, 90 s) for electrophoresis caused association of the two polypeptides into large aggregates. It is concluded that these two polypeptides, 43,000 and 27,000, are valid structural or functional components of Photosystem II pigment-protein complex. PMID:444494

  16. 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.

  17. Focal adhesion proteins connect IgE receptors to the cytoskeleton as revealed by micropatterned ligand arrays

    PubMed Central

    Torres, Alexis J.; Vasudevan, Lavanya; Holowka, David; Baird, Barbara A.

    2008-01-01

    Patterned surfaces that present specific ligands in spatially defined arrays are used to examine structural linkages between clustered IgE receptors (IgE-FcεRI) and the cytoskeleton in rat basophilic leukemia (RBL) mast cells. We showed with fluorescence microscopy that cytoskeletal F-actin concentrates in the same regions as cell surface IgE-FcεRI that bind to the micrometer-size patterned ligands. However, the proteins mediating these cytoskeletal connections and their functional relevance were not known. We now show that whereas the adaptor proteins ezrin and moesin do not detectably concentrate with the array of clustered IgE-FcεRI, focal adhesion proteins vinculin, paxillin, and talin, which are known to link F-actin with integrins, accumulate in these regions on the same time scale as F-actin. Moreover, colocalization of these focal adhesion proteins with clustered IgE-FcεRI is enhanced after addition of fibronectin-RGD peptides. Significantly, the most prominent rat basophilic leukemia cell integrin (α5) avoids the patterned regions occupied by the ligands and associates preferentially with exposed regions of the silicon substrate. Thus, spatial separation provided by the patterned surface reveals that particular focal adhesion proteins, which connect to the actin cytoskeleton, associate with ligand-cross-linked IgE-FcεRI, independently of integrins. We investigated the functional role of one of these proteins, paxillin, in IgE-FcεRI-mediated signaling by using small interfering RNA. From these results, we determine that paxillin reduces stimulated phosphorylation of the FcεRI β subunit but enhances stimulated Ca2+ release from intracellular stores. The results suggest that paxillin associated with clustered IgE-FcεRI has a net positive effect on FcεRI signaling. PMID:19004813

  18. Sporulation Phosporelay Proteins And Their Complexes: Crystallographic Characterization

    SciTech Connect

    Varughese, K.I.; Zhao, H.; Veldore, V.H.; Zapf, J.

    2009-06-04

    Bacteria use two-component systems to adapt to changes in environmental conditions. In response to deteriorating conditions of growth, certain types of bacteria form spores instead of proceeding with cell division. The formation of spores is controlled by an expanded version of two-component systems called the phosphorelay. The phosphorelay comprises a primary kinase that receives the signal/stimulus and undergoes autophosphorylation, followed by two intermediate messengers that regulate the flow of the phosphoryl group to the ultimate response regulator/transcription factor. Sporulation is initiated when the level of phosphorylation of the transcription factor reaches a critical point. This chapter describes efforts to understand the mechanism of initiation of sporulation at the molecular level using X-ray crystallography as a tool. Structural analyses of individual members, as well as their complexes, provide insight into the mechanism of phosphoryl transfer and the origin of specificity in signal transduction.

  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. MyRIP anchors protein kinase A to the exocyst complex.

    PubMed

    Goehring, April S; Pedroja, Benjamin S; Hinke, Simon A; Langeberg, Lorene K; Scott, John D

    2007-11-01

    The movement of signal transduction enzymes in and out of multi-protein complexes coordinates the spatial and temporal resolution of cellular events. Anchoring and scaffolding proteins are key to this process because they sequester protein kinases and phosphatases with a subset of their preferred substrates. The protein kinase A-anchoring family of proteins (AKAPs), which target the cAMP-dependent protein kinase (PKA) and other enzymes to defined subcellular microenvironments, represent a well studied group of these signal-organizing molecules. In this report we demonstrate that the Rab27a GTPase effector protein MyRIP is a member of the AKAP family. The zebrafish homolog of MyRIP (Ze-AKAP2) was initially detected in a two-hybrid screen for AKAPs. A combination of biochemical, cell-based, and immunofluorescence approaches demonstrate that the mouse MyRIP ortholog targets the type II PKA holoenzyme via an atypical mechanism to a specific perinuclear region of insulin-secreting cells. Similar approaches show that MyRIP interacts with the Sec6 and Sec8 components of the exocyst complex, an evolutionarily conserved protein unit that controls protein trafficking and exocytosis. These data indicate that MyRIP functions as a scaffolding protein that links PKA to components of the exocytosis machinery. PMID:17827149

  1. 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

  2. A γ-cyclodextrin duplex connected with two disulfide bonds: synthesis, structure and inclusion complexes.

    PubMed

    Volkov, Sergey; Kumprecht, Lukáš; Buděšínský, Miloš; Lepšík, Martin; Dušek, Michal; Kraus, Tomáš

    2015-03-14

    Per(2,3,6-tri-O-benzyl)-γ-cyclodextrin was debenzylated by DIBAL-H to produce a mixture of C6(I),C6(IV) and C6(I),C6(V) isomeric diols, which were separated and isolated. The C2-symmetrical C6(I),C6(V) diol was transformed into dithiol and dimerized to produce a γ-cyclodextrin duplex structure. A crystal structure revealed tubular cavity whose peripheries are slightly elliptically distorted. The solvent accessible volume of the cavity of the γ-CD duplex is about 740 Å(3). Due to this large inner space the duplex forms very stable inclusion complexes with steroids; bile acids examined in this study show binding affinities to the γ-cyclodextrin duplex in the range of 5.3 × 10(7) M(-1)-1.9 × 10(8) M(-1). PMID:25616110

  3. 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

  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. Alpha-ketoglutarate dehydrogenase complex-dependent succinylation of proteins in neurons and neuronal cell lines.

    PubMed

    Gibson, Gary E; Xu, Hui; Chen, Huan-Lian; Chen, Wei; Denton, Travis T; Zhang, Sheng

    2015-07-01

    Reversible post-translation modifications of proteins are common in all cells and appear to regulate many processes. Nevertheless, the enzyme(s) responsible for the alterations and the significance of the modification are largely unknown. Succinylation of proteins occurs and causes large changes in the structure of proteins; however, the source of the succinyl groups, the targets, and the consequences of these modifications on other proteins remain unknown. These studies focused on succinylation of mitochondrial proteins. The results demonstrate that the α-ketoglutarate dehydrogenase complex (KGDHC) can serve as a trans-succinylase that mediates succinylation in an α-ketoglutarate-dependent manner. Inhibition of KGDHC reduced succinylation of both cytosolic and mitochondrial proteins in cultured neurons and in a neuronal cell line. Purified KGDHC can succinylate multiple proteins including other enzymes of the tricarboxylic acid cycle leading to modification of their activity. Inhibition of KGDHC also modifies acetylation by modifying the pyruvate dehydrogenase complex. The much greater effectiveness of KGDHC than succinyl-CoA suggests that the catalysis owing to the E2k succinyltransferase is important. Succinylation appears to be a major signaling system and it can be mediated by KGDHC. Reversible post-translation modifications of proteins are common and may regulate many processes. Succinylation of proteins occurs and causes large changes in the structure of proteins. However, the source of the succinyl groups, the targets, and the consequences of these modifications on other proteins remains unknown. The results demonstrate that the mitochondrial α-ketoglutarate dehydrogenase complex (KGDHC) can succinylate multiple mitochondrial proteins and alter their function. Succinylation appears to be a major signaling system and it can be mediated by KGDHC. PMID:25772995

  6. Modified bimolecular fluorescence complementation assay to study the inhibition of transcription complex formation by JAZ proteins.

    PubMed

    Qi, Tiancong; Song, Susheng; Xie, Daoxin

    2013-01-01

    The jasmonate (JA) ZIM-domain (JAZ) proteins of Arabidopsis thaliana repress JA signaling and negatively regulate the JA responses. Recently, JAZ proteins have been found to inhibit the transcriptional function of several transcription factors, among which the basic helix-loop-helix (bHLH) (GLABRA3 [GL3], ENHANCER OF GLABRA3 [EGL3], and TRANSPARENT TESTA8 [TT8]) and R2R3-MYB (GL1 and MYB75) that can interact with each other to form bHLH-MYB complexes and further control gene expression. The bimolecular fluorescence complementation (BiFC) assay is a widely used technique to study protein-protein interactions in living cells. Here we describe a modified BiFC experimental procedure to study the inhibition of the formation of the bHLH (GL3)-MYB (GL1) complex by JAZ proteins. PMID:23615997

  7. Biopolymer nanoparticles from heat-treated electrostatic protein-polysaccharide complexes: factors affecting particle characteristics.

    PubMed

    Jones, Owen Griffith; McClements, David Julian

    2010-03-01

    Biopolymer nanoparticles can be formed by heating globular protein-ionic polysaccharide electrostatic complexes above the thermal denaturation temperature of the protein. This study examined how the size and concentration of biopolymer particles formed by heating beta-lactoglobulin-pectin complexes could be manipulated by controlling preparation conditions: pH, ionic strength, protein concentration, holding time, and holding temperature. Biopolymer particle size and concentration increased with increasing holding time (0 to 30 min), decreasing holding temperature (90 to 70 degrees C), increasing protein concentration (0 to 2 wt/wt%), increasing pH (4.5 to 5), and increasing salt concentration (0 to 50 mol/kg). The influence of these factors on biopolymer particle size was attributed to their impact on protein-polysaccharide interactions, and on the kinetics of nucleation and particle growth. The knowledge gained from this study will facilitate the rational design of biopolymer particles with specific physicochemical and functional attributes. PMID:20492252

  8. Protein degradation corrects for imbalanced subunit stoichiometry in OST complex assembly

    PubMed Central

    Mueller, Susanne; Wahlander, Asa; Selevsek, Nathalie; Otto, Claudia; Ngwa, Elsy Mankah; Poljak, Kristina; Frey, Alexander D.; Aebi, Markus; Gauss, Robert

    2015-01-01

    Protein degradation is essential for cellular homeostasis. We developed a sensitive approach to examining protein degradation rates in Saccharomyces cerevisiae by coupling a SILAC approach to selected reaction monitoring (SRM) mass spectrometry. Combined with genetic tools, this analysis made it possible to study the assembly of the oligosaccharyl transferase complex. The ER-associated degradation machinery compensated for disturbed homeostasis of complex components by degradation of subunits in excess. On a larger scale, protein degradation in the ER was found to be a minor factor in the regulation of protein homeostasis in exponentially growing cells, but ERAD became relevant when the gene dosage was affected, as demonstrated in heterozygous diploid cells. Hence the alleviation of fitness defects due to abnormal gene copy numbers might be an important function of protein degradation. PMID:25995378

  9. Crystal Structures of the Tryptophan Repressor binding Protein WrbA and complexes with Flavin Mononucleotide

    SciTech Connect

    Gorman,J.; Shapiro, L.

    2005-01-01

    The tryptophan repressor binding protein WrbA binds to the tryptophan repressor protein TrpR. Although the biological role of WrbA remains unclear, it has been proposed to function in enhancing the stability of TrpR-DNA complexes. Sequence database analysis has identified WrbA as a founding member of a flavodoxin-like family of proteins. Here we present crystal structures of WrbA from Deinococcus radiodurans and Pseudomonas aeruginosa and their complexes with flavin mononucleotide. The protomer structure is similar to that of previously determined long-chain flavodoxins; however, each contains a conserved inserted region unique to the WrbA family. Interestingly, each WrbA protein forms a homotetramer with 222 symmetry, unique among flavodoxin-like proteins, in which each protomer binds one flavin mononucleotide cofactor molecule.

  10. Acute phase proteins in cattle after exposure to complex stress.<