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Sample records for interactive protein manipulation

  1. Interactive protein manipulation

    SciTech Connect

    SNCrivelli@lbl.gov

    2003-07-01

    We describe an interactive visualization and modeling program for the creation of protein structures ''from scratch''. The input to our program is an amino acid sequence -decoded from a gene- and a sequence of predicted secondary structure types for each amino acid-provided by external structure prediction programs. Our program can be used in the set-up phase of a protein structure prediction process; the structures created with it serve as input for a subsequent global internal energy minimization, or another method of protein structure prediction. Our program supports basic visualization methods for protein structures, interactive manipulation based on inverse kinematics, and visualization guides to aid a user in creating ''good'' initial structures.

  2. A Tool for Interactive Protein Manipulation

    Energy Science and Technology Software Center (ESTSC)

    2005-03-28

    ProteinShop is a graphical environment that facilitates a solution to the protein prediction problem through a combination of unique features and capabilities. These include: 1. Helping researchers automatically generate 3D protein structures from scratcW by using the sequence of amino acids and secondary structure specifications as input. 2. Enabling users to apply their accumulated biochemical knowledge and intuition during the interactive manipulation of structures. 3. FacIlitating interactive comparison and analysis of alternative structures through visualizationmore » of free energy computed during modeling. 4. Accelerating discovery of low-energy configurations by applying local optimizations plug-in to user-selected protein structures. ProteinShop v.2.0 includes the following new features: - Visualizes multiple-domain structures - Automatically creates a user-specified number of beta-sheet configurations - Provides the interface and the libraries for energy visualization and local minimization of protein structures - Reads standard POB files without previous editing« less

  3. ProteinShop: A tool for interactive protein manipulation and steering

    SciTech Connect

    Crivelli, Silvia; Kreylos, Oliver; Max, Nelson; Hamann, Bernd; Bethel, Wes

    2004-05-25

    We describe ProteinShop, a new visualization tool that streamlines and simplifies the process of determining optimal protein folds. ProteinShop may be used at different stages of a protein structure prediction process. First, it can create protein configurations containing secondary structures specified by the user. Second, it can interactively manipulate protein fragments to achieve desired folds by adjusting the dihedral angles of selected coil regions using an Inverse Kinematics method. Last, it serves as a visual framework to monitor and steer a protein structure prediction process that may be running on a remote machine. ProteinShop was used to create initial configurations for a protein structure prediction method developed by a team that competed in CASP5. ProteinShop's use accelerated the process of generating initial configurations, reducing the time required from days to hours. This paper describes the structure of ProteinShop and discusses its main features.

  4. Manipulating Fatty Acid Biosynthesis in Microalgae for Biofuel through Protein-Protein Interactions

    PubMed Central

    Blatti, Jillian L.; Beld, Joris; Behnke, Craig A.; Mendez, Michael; Mayfield, Stephen P.; Burkart, Michael D.

    2012-01-01

    Microalgae are a promising feedstock for renewable fuels, and algal metabolic engineering can lead to crop improvement, thus accelerating the development of commercially viable biodiesel production from algae biomass. We demonstrate that protein-protein interactions between the fatty acid acyl carrier protein (ACP) and thioesterase (TE) govern fatty acid hydrolysis within the algal chloroplast. Using green microalga Chlamydomonas reinhardtii (Cr) as a model, a structural simulation of docking CrACP to CrTE identifies a protein-protein recognition surface between the two domains. A virtual screen reveals plant TEs with similar in silico binding to CrACP. Employing an activity-based crosslinking probe designed to selectively trap transient protein-protein interactions between the TE and ACP, we demonstrate in vitro that CrTE must functionally interact with CrACP to release fatty acids, while TEs of vascular plants show no mechanistic crosslinking to CrACP. This is recapitulated in vivo, where overproduction of the endogenous CrTE increased levels of short-chain fatty acids and engineering plant TEs into the C. reinhardtii chloroplast did not alter the fatty acid profile. These findings highlight the critical role of protein-protein interactions in manipulating fatty acid biosynthesis for algae biofuel engineering as illuminated by activity-based probes. PMID:23028438

  5. Potato leafroll virus structural proteins manipulate overlapping, yet distinct protein interaction networks during infection.

    PubMed

    DeBlasio, Stacy L; Johnson, Richard; Sweeney, Michelle M; Karasev, Alexander; Gray, Stewart M; MacCoss, Michael J; Cilia, Michelle

    2015-06-01

    Potato leafroll virus (PLRV) produces a readthrough protein (RTP) via translational readthrough of the coat protein amber stop codon. The RTP functions as a structural component of the virion and as a nonincorporated protein in concert with numerous insect and plant proteins to regulate virus movement/transmission and tissue tropism. Affinity purification coupled to quantitative MS was used to generate protein interaction networks for a PLRV mutant that is unable to produce the read through domain (RTD) and compared to the known wild-type PLRV protein interaction network. By quantifying differences in the protein interaction networks, we identified four distinct classes of PLRV-plant interactions: those plant and nonstructural viral proteins interacting with assembled coat protein (category I); plant proteins in complex with both coat protein and RTD (category II); plant proteins in complex with the RTD (category III); and plant proteins that had higher affinity for virions lacking the RTD (category IV). Proteins identified as interacting with the RTD are potential candidates for regulating viral processes that are mediated by the RTP such as phloem retention and systemic movement and can potentially be useful targets for the development of strategies to prevent infection and/or viral transmission of Luteoviridae species that infect important crop species. PMID:25787689

  6. Pharmacological manipulation of transcription factor protein-protein interactions: opportunities and obstacles.

    PubMed

    Fontaine, Frank; Overman, Jeroen; François, Mathias

    2015-01-01

    Much research on transcription factor biology and their genetic pathways has been undertaken over the last 30 years, especially in the field of developmental biology and cancer. Yet, very little is known about the molecular modalities of highly dynamic interactions between transcription factors, genomic DNA, and protein partners. Methodological breakthroughs such as RNA-seq (RNA-sequencing), ChIP-seq (chromatin immunoprecipitation sequencing), RIME (rapid immunoprecipitation mass spectrometry of endogenous proteins), and single-molecule imaging will dramatically accelerate the discovery rate of their molecular mode of action in the next few years. From a pharmacological viewpoint, conventional methods used to target transcription factor activity with molecules mimicking endogenous ligands fail to achieve high specificity and are limited by a lack of identification of new molecular targets. Protein-protein interactions are likely to represent one of the next major classes of therapeutic targets. Transcription factors, known to act mostly via protein-protein interaction, may well be at the forefront of this type of drug development. One hurdle in this field remains the difficulty to collate structural data into meaningful information for rational drug design. Another hurdle is the lack of chemical libraries meeting the structural requirements of protein-protein interaction disruption. As more attempts at modulating transcription factor activity are undertaken, valuable knowledge will be accumulated on the modality of action required to modulate transcription and how these findings can be applied to developing transcription factor drugs. Key discoveries will spawn into new therapeutic approaches not only as anticancer targets but also for other indications, such as those with an inflammatory component including neurodegenerative disorders, diabetes, and chronic liver and kidney diseases. PMID:25848531

  7. Energetic Manipulation of Chloroplast Protein Import and the Use of Chemical Cross-Linkers to Map Protein–Protein Interactions

    PubMed Central

    Inoue, Hitoshi; Wang, Fei; Inaba, Takehito; Schnell, Danny J.

    2014-01-01

    Most chloroplast proteins are synthesized in the cytosol as preproteins with N-terminal cleavable transit peptides and are imported into the organelle through the TOC–TIC translocon system. Import involves a complex set of recognition and membrane translocation steps that ensure the fidelity and unidirectional transport of the polypeptide across the double-membrane chloroplast envelope. To understand the mechanism of import, the molecular interactions and energetics of each step must be defined. Here, we describe the methods for capturing intermediates in the import process through the manipulation of the energy state of chloroplasts, and the use of two different chemical cross-linking approaches to examine the molecular interactions that mediate the import process and to assess the assembly state of the translocons. These approaches can be employed to identify sequential protein–protein interactions, and thereby dissect the pathway and roles of import components during protein import into chloroplasts. PMID:21822846

  8. The Potato leafroll virus structural proteins manipulate overlapping, yet distinct protein interaction networks during infection

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Potato leafroll virus (PLRV) produces a readthrough protein (RTP) via translational readthrough of the coat protein amber stop codon. The RTP functions as a structural component of the virion and as a non-incorporated protein in concert with numerous insect and plant proteins to regulate virus movem...

  9. Manipulating and Visualizing Proteins

    SciTech Connect

    Simon, Horst D.

    2003-12-05

    ProteinShop Gives Researchers a Hands-On Tool for Manipulating, Visualizing Protein Structures. The Human Genome Project and other biological research efforts are creating an avalanche of new data about the chemical makeup and genetic codes of living organisms. But in order to make sense of this raw data, researchers need software tools which let them explore and model data in a more intuitive fashion. With this in mind, researchers at Lawrence Berkeley National Laboratory and the University of California, Davis, have developed ProteinShop, a visualization and modeling program which allows researchers to manipulate protein structures with pinpoint control, guided in large part by their own biological and experimental instincts. Biologists have spent the last half century trying to unravel the ''protein folding problem,'' which refers to the way chains of amino acids physically fold themselves into three-dimensional proteins. This final shape, which resembles a crumpled ribbon or piece of origami, is what determines how the protein functions and translates genetic information. Understanding and modeling this geometrically complex formation is no easy matter. ProteinShop takes a given sequence of amino acids and uses visualization guides to help generate predictions about the secondary structures, identifying alpha helices and flat beta strands, and the coil regions that bind them. Once secondary structures are in place, researchers can twist and turn these pre-configurations until they come up with a number of possible tertiary structure conformations. In turn, these are fed into a computationally intensive optimization procedure that tries to find the final, three-dimensional protein structure. Most importantly, ProteinShop allows users to add human knowledge and intuition to the protein structure prediction process, thus bypassing bad configurations that would otherwise be fruitless for optimization. This saves compute cycles and accelerates the entire process, so

  10. Combining Single-molecule Manipulation and Imaging for the Study of Protein-DNA Interactions

    PubMed Central

    Monico, Carina; Belcastro, Gionata; Vanzi, Francesco; Pavone, Francesco S.; Capitanio, Marco

    2014-01-01

    The paper describes the combination of optical tweezers and single molecule fluorescence detection for the study of protein-DNA interaction. The method offers the opportunity of investigating interactions occurring in solution (thus avoiding problems due to closeby surfaces as in other single molecule methods), controlling the DNA extension and tracking interaction dynamics as a function of both mechanical parameters and DNA sequence. The methods for establishing successful optical trapping and nanometer localization of single molecules are illustrated. We illustrate the experimental conditions allowing the study of interaction of lactose repressor (lacI), labeled with Atto532, with a DNA molecule containing specific target sequences (operators) for LacI binding. The method allows the observation of specific interactions at the operators, as well as one-dimensional diffusion of the protein during the process of target search. The method is broadly applicable to the study of protein-DNA interactions but also to molecular motors, where control of the tension applied to the partner track polymer (for example actin or microtubules) is desirable. PMID:25226304

  11. Exploring protein-DNA interactions in 3D using in situ construction, manipulation, and visualization of individual DNA-dumbbells with optical traps, microfluidics, and fluorescence microscopy

    PubMed Central

    Forget, Anthony L.; Dombrowski, Christopher C.; Amitani, Ichiro; Kowalczykowski, Stephen C.

    2015-01-01

    In this Protocol, we describe a procedure to generate ‘DNA-dumbbells’ — single molecules of DNA with a microscopic bead attached at each end — and techniques for manipulating individual DNA-dumbbells. We also detail the design and fabrication of a microfluidic device (flow cell) used in conjunction with dual optical trapping to manipulate DNA-dumbbells and to visualize individual protein–DNA complexes by single-molecule epifluorescence microscopy. Our design of the flow cell enables the rapid movement of trapped molecules between laminar flow channels and a flow-free ‘reservoir’. The reservoir provides the means to examine formation of DNA–protein complexes in solution in the absence of external flow forces, while still maintaining a predetermined end-to-end extension of the DNA. These features facilitate examination of the role of three-dimensional DNA conformation and dynamics in protein–DNA interactions. Preparation of flow cells and reagents requires two days each; in situ DNA-dumbbell assembly and imaging of single protein–DNA complexes requires another day. PMID:23411634

  12. Interactive digital image manipulation system

    NASA Technical Reports Server (NTRS)

    Henze, J.; Dezur, R.

    1975-01-01

    The system is designed for manipulation, analysis, interpretation, and processing of a wide variety of image data. LANDSAT (ERTS) and other data in digital form can be input directly into the system. Photographic prints and transparencies are first converted to digital form with an on-line high-resolution microdensitometer. The system is implemented on a Hewlett-Packard 3000 computer with 128 K bytes of core memory and a 47.5 megabyte disk. It includes a true color display monitor, with processing memories, graphics overlays, and a movable cursor. Image data formats are flexible so that there is no restriction to a given set of remote sensors. Conversion between data types is available to provide a basis for comparison of the various data. Multispectral data is fully supported, and there is no restriction on the number of dimensions. In this way multispectral data collected at more than one point in time may simply be treated as a data collected with twice (three times, etc.) the number of sensors. There are various libraries of functions available to the user: processing functions, display functions, system functions, and earth resources applications functions.

  13. A 3D interactive optical manipulation platform

    NASA Astrophysics Data System (ADS)

    Glückstad, Jesper; Rodrigo, Peter J.; Nielson, Ivan P.

    2005-12-01

    Three-dimensional light structures can be created by modulating the spatial phase and polarization properties of the laser light. A particularly promising technique is the Generalized Phase Contrast (GPC) method invented and patented at Riso National Laboratory. Based on the combination of programmable spatial light modulator devices and an advanced graphical user-interface the GPC method enables real-time, interactive and arbitrary control over the dynamics and geometry of synthesized light patterns. Recent experiments have shown that GPC-driven micro-manipulation provides a unique technology platform for fully user-guided assembly of a plurality of particles in a plane, control of particle stacking along the beam axis, manipulation of multiple hollow beads, and the organization of living cells into three-dimensional colloidal structures. These demonstrations illustrate that GPC-driven micro-manipulation can be utilized not only for the improved synthesis of functional microstructures but also for non-contact and parallel actuation crucial for sophisticated opto- and micro-fluidic based lab-on-a-chip systems.

  14. Manipulator interactive design with interconnected flexible elements

    NASA Technical Reports Server (NTRS)

    Singh, R. P.; Likins, P. W.

    1983-01-01

    This paper describes the development of an analysis tool for the interactive design of control systems for manipulators and similar electro-mechanical systems amenable to representation as structures in a topological chain. The chain consists of a series of elastic bodies subject to small deformations and arbitrary displacements. The bodies are connected by hinges which permit kinematic constraints, control, or relative motion with six degrees of freedom. The equations of motion for the chain configuration are derived via Kane's method, extended for application to interconnected flexible bodies with time-varying boundary conditions. A corresponding set of modal coordinates has been selected. The motion equations are imbedded within a simulation that transforms the vector-dyadic equations into scalar form for numerical integration. The simulation also includes a linear, time-invariant controler specified in transfer function format and a set of sensors and actuators that interface between the structure and controller. The simulation is driven by an interactive set-up program resulting in an easy-to-use analysis tool.

  15. PIC: Protein Interactions Calculator.

    PubMed

    Tina, K G; Bhadra, R; Srinivasan, N

    2007-07-01

    Interactions within a protein structure and interactions between proteins in an assembly are essential considerations in understanding molecular basis of stability and functions of proteins and their complexes. There are several weak and strong interactions that render stability to a protein structure or an assembly. Protein Interactions Calculator (PIC) is a server which, given the coordinate set of 3D structure of a protein or an assembly, computes various interactions such as disulphide bonds, interactions between hydrophobic residues, ionic interactions, hydrogen bonds, aromatic-aromatic interactions, aromatic-sulphur interactions and cation-pi interactions within a protein or between proteins in a complex. Interactions are calculated on the basis of standard, published criteria. The identified interactions between residues can be visualized using a RasMol and Jmol interface. The advantage with PIC server is the easy availability of inter-residue interaction calculations in a single site. It also determines the accessible surface area and residue-depth, which is the distance of a residue from the surface of the protein. User can also recognize specific kind of interactions, such as apolar-apolar residue interactions or ionic interactions, that are formed between buried or exposed residues or near the surface or deep inside. PMID:17584791

  16. Bacteriophage protein-protein interactions.

    PubMed

    Häuser, Roman; Blasche, Sonja; Dokland, Terje; Haggård-Ljungquist, Elisabeth; von Brunn, Albrecht; Salas, Margarita; Casjens, Sherwood; Molineux, Ian; Uetz, Peter

    2012-01-01

    Bacteriophages T7, λ, P22, and P2/P4 (from Escherichia coli), as well as ϕ29 (from Bacillus subtilis), are among the best-studied bacterial viruses. This chapter summarizes published protein interaction data of intraviral protein interactions, as well as known phage-host protein interactions of these phages retrieved from the literature. We also review the published results of comprehensive protein interaction analyses of Pneumococcus phages Dp-1 and Cp-1, as well as coliphages λ and T7. For example, the ≈55 proteins encoded by the T7 genome are connected by ≈43 interactions with another ≈15 between the phage and its host. The chapter compiles published interactions for the well-studied phages λ (33 intra-phage/22 phage-host), P22 (38/9), P2/P4 (14/3), and ϕ29 (20/2). We discuss whether different interaction patterns reflect different phage lifestyles or whether they may be artifacts of sampling. Phages that infect the same host can interact with different host target proteins, as exemplified by E. coli phage λ and T7. Despite decades of intensive investigation, only a fraction of these phage interactomes are known. Technical limitations and a lack of depth in many studies explain the gaps in our knowledge. Strategies to complete current interactome maps are described. Although limited space precludes detailed overviews of phage molecular biology, this compilation will allow future studies to put interaction data into the context of phage biology. PMID:22748812

  17. Interactive Streamline Exploration and Manipulation Using Deformation

    SciTech Connect

    Tong, Xin; Chen, Chun-Ming; Shen, Han-Wei; Wong, Pak C.

    2015-01-12

    Occlusion presents a major challenge in visualizing three-dimensional flow fields with streamlines. Displaying too many streamlines at once makes it difficult to locate interesting regions, but displaying too few streamlines risks missing important features. A more ideal streamline exploration model is to allow the viewer to freely move across the field that has been populated with interesting streamlines and pull away the streamlines that cause occlusion so that the viewer can inspect the hidden ones in detail. In this paper, we present a streamline deformation algorithm that supports such user-driven interaction with three-dimensional flow fields. We define a view-dependent focus+context technique that moves the streamlines occluding the focus area using a novel displacement model. To preserve the context surrounding the user-chosen focus area, we propose two shape models to define the transition zone for the surrounding streamlines, and the displacement of the contextual streamlines is solved interactively with a goal of preserving their shapes as much as possible. Based on our deformation model, we design an interactive streamline exploration tool using a lens metaphor. Our system runs interactively so that users can move their focus and examine the flow field freely.

  18. MONA – Interactive manipulation of molecule collections

    PubMed Central

    2013-01-01

    Working with small‐molecule datasets is a routine task for cheminformaticians and chemists. The analysis and comparison of vendor catalogues and the compilation of promising candidates as starting points for screening campaigns are but a few very common applications. The workflows applied for this purpose usually consist of multiple basic cheminformatics tasks such as checking for duplicates or filtering by physico‐chemical properties. Pipelining tools allow to create and change such workflows without much effort, but usually do not support interventions once the pipeline has been started. In many contexts, however, the best suited workflow is not known in advance, thus making it necessary to take the results of the previous steps into consideration before proceeding. To support intuition‐driven processing of compound collections, we developed MONA, an interactive tool that has been designed to prepare and visualize large small‐molecule datasets. Using an SQL database common cheminformatics tasks such as analysis and filtering can be performed interactively with various methods for visual support. Great care was taken in creating a simple, intuitive user interface which can be instantly used without any setup steps. MONA combines the interactivity of molecule database systems with the simplicity of pipelining tools, thus enabling the case‐to‐case application of chemistry expert knowledge. The current version is available free of charge for academic use and can be downloaded at http://www.zbh.uni‐hamburg.de/mona. PMID:23985157

  19. Complex interactions of multiple aquatic consumers: an experimental mesocosm manipulation

    USGS Publications Warehouse

    Richardson, William B.; Threlkeld, Stephen T.

    1993-01-01

    In 7-m3 outdoor tanks filled with lake water, the presence/absence of omnivorous young-of-the- year Micropterus salmoides), zooplanktivorous Menidia beryllina , and herbivorous larval Hyla chrysocelis was experimentally manipulated. A cross-classified design was used to assess the interactive effects of these vertebrate consumers on the experimental food webs. The primary effects of the experimental manipulations on food web components were two- and three-way interactions in which the effect of a given treatment was dependent on the presence of another treatment. Results suggest that the addition or removal of consumers may not cause linear, additive changes in food webs.

  20. Two-Dimensional Heterojunctions from Nonlocal Manipulations of the Interactions.

    PubMed

    Rösner, M; Steinke, C; Lorke, M; Gies, C; Jahnke, F; Wehling, T O

    2016-04-13

    We propose to create lateral heterojunctions in two-dimensional materials based on nonlocal manipulations of the Coulomb interaction using structured dielectric environments. By means of ab initio calculations for MoS2 as well as generic semiconductor models, we show that the Coulomb interaction-induced self-energy corrections in real space are sufficiently nonlocal to be manipulated externally, but still local enough to induce spatially sharp interfaces within a single homogeneous monolayer to form heterojunctions. We find a type-II heterojunction band scheme promoted by a laterally structured dielectric environment, which exhibits a sharp band gap crossover within less than 5 unit cells. PMID:26918626

  1. Drugging Membrane Protein Interactions.

    PubMed

    Yin, Hang; Flynn, Aaron D

    2016-07-11

    The majority of therapeutics target membrane proteins, accessible on the surface of cells, to alter cellular signaling. Cells use membrane proteins to transduce signals into cells, transport ions and molecules, bind cells to a surface or substrate, and catalyze reactions. Newly devised technologies allow us to drug conventionally "undruggable" regions of membrane proteins, enabling modulation of protein-protein, protein-lipid, and protein-nucleic acid interactions. In this review, we survey the state of the art of high-throughput screening and rational design in drug discovery, and we evaluate the advances in biological understanding and technological capacity that will drive pharmacotherapy forward against unorthodox membrane protein targets. PMID:26863923

  2. Cellulose synthase interacting protein

    PubMed Central

    Somerville, Chris

    2010-01-01

    Cellulose is the most abundant biopolymer on earth. The great abundance of cellulose places it at the forefront as a primary source of biomass for renewable biofuels. However, the knowledge of how plant cells make cellulose remains very rudimentary. Cellulose microfibrils are synthesized at the plasma membrane by hexameric protein complexes, also known as cellulose synthase complexes. The only known components of cellulose synthase complexes are cellulose synthase (CESA) proteins until the recent identification of a novel component. CSI1, which encodes CESA interacting protein 1 (CSI1) in Arabidopsis. CSI1, as the first non-CESA proteins associated with cellulose synthase complexes, opens up many opportunities. PMID:21150290

  3. Manipulation in close relationships: five personality factors in interactional context.

    PubMed

    Buss, D M

    1992-06-01

    This research had three basic goals: (a) to identify manipulation tactics used in close relationships; (b) to document empirically the degree of generality and specificity of tactical deployment across relationship types (mates, friends, parents); and (c) to identify links between five major personality dimensions and the usage of manipulation tactics. Twelve manipulation tactics were identified through separate factor analyses of two instruments based on different data sources: Charm, Reason, Coercion, Silent Treatment, Debasement, and Regression (replicating Buss et al., 1987), and Responsibility Invocation, Reciprocity, Monetary Reward, Pleasure Induction, Social Comparison, and Hardball (an amalgam of threats, lies, and violence). The Big Five personality factors were assessed through three separate data sources: self-report, spouse report, and two independent interviewers. Personality factors showed coherent links with tactics, including Surgency (Coercion, Responsibility, Invocation), Desurgency (Debasement), Agreeableness (Pleasure Induction), Disagreeableness (Coercion), Conscientiousness (Reason), Emotional Instability (Regression), and Intellect-Openness (Reason). Discussion focuses on the consequences of the five personality factors for social interaction in close relationships. PMID:1635051

  4. Drugging Membrane Protein Interactions

    PubMed Central

    Yin, Hang; Flynn, Aaron D.

    2016-01-01

    The majority of therapeutics target membrane proteins, accessible on the surface of cells, to alter cellular signaling. Cells use membrane proteins to transduce signals into cells, transport ions and molecules, bind the cell to a surface or substrate, and catalyze reactions. Newly devised technologies allow us to drug conventionally “undruggable” regions of membrane proteins, enabling modulation of protein–protein, protein–lipid, and protein–nucleic acid interactions. In this review, we survey the state of the art in high-throughput screening and rational design in drug discovery, and we evaluate the advances in biological understanding and technological capacity that will drive pharmacotherapy forward against unorthodox membrane protein targets. PMID:26863923

  5. Cotton and Protein Interactions

    SciTech Connect

    Goheen, Steven C.; Edwards, J. V.; Rayburn, Alfred R.; Gaither, Kari A.; Castro, Nathan J.

    2006-06-30

    The adsorbent properties of important wound fluid proteins and cotton cellulose are reviewed. This review focuses on the adsorption of albumin to cotton-based wound dressings and some chemically modified derivatives targeted for chronic wounds. Adsorption of elastase in the presence of albumin was examined as a model to understand the interactive properties of these wound fluid components with cotton fibers. In the chronic non-healing wound, elastase appears to be over-expressed, and it digests tissue and growth factors, interfering with the normal healing process. Albumin is the most prevalent protein in wound fluid, and in highly to moderately exudative wounds, it may bind significantly to the fibers of wound dressings. Thus, the relative binding properties of both elastase and albumin to wound dressing fibers are of interest in the design of more effective wound dressings. The present work examines the binding of albumin to two different derivatives of cotton, and quantifies the elastase binding to the same derivatives following exposure of albumin to the fiber surface. An HPLC adsorption technique was employed coupled with a colorimetric enzyme assay to quantify the relative binding properties of albumin and elastase to cotton. The results of wound protein binding are discussed in relation to the porosity and surface chemistry interactions of cotton and wound proteins. Studies are directed to understanding the implications of protein adsorption phenomena in terms of fiber-protein models that have implications for rationally designing dressings for chronic wounds.

  6. Actin Cytoskeleton Manipulation by Effector Proteins Secreted by Diarrheagenic Escherichia coli Pathotypes

    PubMed Central

    Navarro-Garcia, Fernando; Serapio-Palacios, Antonio; Ugalde-Silva, Paul; Tapia-Pastrana, Gabriela; Chavez-Dueñas, Lucia

    2013-01-01

    The actin cytoskeleton is a dynamic structure necessary for cell and tissue organization, including the maintenance of epithelial barriers. Disruption of the epithelial barrier coincides with alterations of the actin cytoskeleton in several disease states. These disruptions primarily affect the paracellular space, which is normally regulated by tight junctions. Thereby, the actin cytoskeleton is a common and recurring target of bacterial virulence factors. In order to manipulate the actin cytoskeleton, bacteria secrete and inject toxins and effectors to hijack the host cell machinery, which interferes with host-cell pathways and with a number of actin binding proteins. An interesting model to study actin manipulation by bacterial effectors is Escherichia coli since due to its genome plasticity it has acquired diverse genetic mobile elements, which allow having different E. coli varieties in one bacterial species. These E. coli pathotypes, including intracellular and extracellular bacteria, interact with epithelial cells, and their interactions depend on a specific combination of virulence factors. In this paper we focus on E. coli effectors that mimic host cell proteins to manipulate the actin cytoskeleton. The study of bacterial effector-cytoskeleton interaction will contribute not only to the comprehension of the molecular causes of infectious diseases but also to increase our knowledge of cell biology. PMID:23509714

  7. 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.

  8. Carotenoid-Protein Interactions

    NASA Astrophysics Data System (ADS)

    Britton, George; Helliwell, John R.

    Chapter 5 shows that the aggregation of carotenoid molecules can have a profound effect on their properties and hence their functioning in biological systems. Another important influence is the interaction between carotenoids and other molecules. The way that interactions of carotenoids with lipid bilayers influence the structure and properties of membranes and membrane-asociated processes is discussed in Chapter 10, and the aggregation of carotenoid molecules within the bilayers in Chapter 5. Of particular importance, though, are interactions between carotenoids and proteins. These allow the hydrophobic carotenoids to be transported, to exist, and to function in an aqueous environment. In some cases they may modify strongly the light-absorption properties and hence the colour and photochemistry of the carotenoids.

  9. Engineering light-matter interaction for emerging optical manipulation applications

    NASA Astrophysics Data System (ADS)

    Qiu, Cheng-Wei; Palima, Darwin; Novitsky, Andrey; Gao, Dongliang; Ding, Weiqiang; Zhukovsky, Sergei V.; Gluckstad, Jesper

    2014-06-01

    In this review, we explore recent trends in optical micromanipulation by engineering light-matter interaction and controlling the mechanical effects of optical fields. One central theme is exploring the rich phenomena beyond the now established precision measurements based on trapping micro beads with tightly focused beams. Novel synthesized beams, exploiting the linear and angular momentum of light, open new possibilities in optical trapping and micromanipulation. Similarly, novel structures are promising to enable new optical micromanipulation modalities. Moreover, an overview of the amazing features of the optics of tractor beams and backward-directed energy fluxes will be presented. Recently the so-called effect of negative propagation of the beams (existence of the backward energy fluxes) has been confirmed for X-waves and Airy beams. In the review, we will also discuss the negative pulling force of structured beams and negative energy fluxes in the vicinity of fibers. The effect is achieved due to the interaction of multipoles or, in another interpretation, the momentum conservation. Both backward-directed Poynting vector and backward optical forces are counter-intuitive and give an insight into new physics and technologies. Exploiting the degrees of freedom in synthesizing novel beams and designed microstructures offer attractive prospects for emerging optical manipulation applications.

  10. Parallel algorithms for interactive manipulation of digital terrain models

    NASA Technical Reports Server (NTRS)

    Davis, E. W.; Mcallister, D. F.; Nagaraj, V.

    1988-01-01

    Interactive three-dimensional graphics applications, such as terrain data representation and manipulation, require extensive arithmetic processing. Massively parallel machines are attractive for this application since they offer high computational rates, and grid connected architectures provide a natural mapping for grid based terrain models. Presented here are algorithms for data movement on the massive parallel processor (MPP) in support of pan and zoom functions over large data grids. It is an extension of earlier work that demonstrated real-time performance of graphics functions on grids that were equal in size to the physical dimensions of the MPP. When the dimensions of a data grid exceed the processing array size, data is packed in the array memory. Windows of the total data grid are interactively selected for processing. Movement of packed data is needed to distribute items across the array for efficient parallel processing. Execution time for data movement was found to exceed that for arithmetic aspects of graphics functions. Performance figures are given for routines written in MPP Pascal.

  11. Nanobiotechnology: protein-nanomaterial interactions.

    PubMed

    Kane, Ravi S; Stroock, Abraham D

    2007-01-01

    We review recent research that involves the interaction of nanomaterials such as nanoparticles, nanowires, and carbon nanotubes with proteins. We begin with a focus on the fundamentals of the structure and function of proteins on nanomaterials. We then review work in three areas that exploit these interactions: (1) sensing, (2) assembly of nanomaterials by proteins and proteins by nanomaterials, and (3) interactions with cells. We conclude with the identification of challenges and opportunities for the future. PMID:17335286

  12. PREFACE: Protein protein interactions: principles and predictions

    NASA Astrophysics Data System (ADS)

    Nussinov, Ruth; Tsai, Chung-Jung

    2005-06-01

    Proteins are the `workhorses' of the cell. Their roles span functions as diverse as being molecular machines and signalling. They carry out catalytic reactions, transport, form viral capsids, traverse membranes and form regulated channels, transmit information from DNA to RNA, making possible the synthesis of new proteins, and they are responsible for the degradation of unnecessary proteins and nucleic acids. They are the vehicles of the immune response and are responsible for viral entry into the cell. Given their importance, considerable effort has been centered on the prediction of protein function. A prime way to do this is through identification of binding partners. If the function of at least one of the components with which the protein interacts is known, that should let us assign its function(s) and the pathway(s) in which it plays a role. This holds since the vast majority of their chores in the living cell involve protein-protein interactions. Hence, through the intricate network of these interactions we can map cellular pathways, their interconnectivities and their dynamic regulation. Their identification is at the heart of functional genomics; their prediction is crucial for drug discovery. Knowledge of the pathway, its topology, length, and dynamics may provide useful information for forecasting side effects. The goal of predicting protein-protein interactions is daunting. Some associations are obligatory, others are continuously forming and dissociating. In principle, from the physical standpoint, any two proteins can interact, but under what conditions and at which strength? The principles of protein-protein interactions are general: the non-covalent interactions of two proteins are largely the outcome of the hydrophobic effect, which drives the interactions. In addition, hydrogen bonds and electrostatic interactions play important roles. Thus, many of the interactions observed in vitro are the outcome of experimental overexpression. Protein disorder

  13. Imaging Protein-protein Interactions in vivo

    PubMed Central

    Seegar, Tom; Barton, William

    2010-01-01

    Protein-protein interactions are a hallmark of all essential cellular processes. However, many of these interactions are transient, or energetically weak, preventing their identification and analysis through traditional biochemical methods such as co-immunoprecipitation. In this regard, the genetically encodable fluorescent proteins (GFP, RFP, etc.) and their associated overlapping fluorescence spectrum have revolutionized our ability to monitor weak interactions in vivo using Förster resonance energy transfer (FRET)1-3. Here, we detail our use of a FRET-based proximity assay for monitoring receptor-receptor interactions on the endothelial cell surface. PMID:20972411

  14. Manipulating forces by interactive separation and circulation control

    NASA Astrophysics Data System (ADS)

    Nagib, Hassan; Reinhard, Paul; Rozier, Paul

    2011-11-01

    Steady, unsteady and intermittent suction and blowing from localized or distributed slots are used to reveal the physical mechanisms and their interaction in order to manipulate (enhance or reduce) the forces on various aerodynamic bodies and surfaces. Performance under ideal inviscid conditions is used as a standard of performance to compare the outcomes to. While high-lift airfoils were part of the focus, flow over humps which lead to large separation zones was also investigated. Surface pressure measurements, wake surveys and surface visualization were utilized over a wide range of operating conditions in the NDF at IIT. Velocities ranged from 20 to 110 m/s (0 . 06 < M < 0 . 31), corresponding to chord Reynolds numbers from 500 , 000 to 3 , 700 , 000 , and included a full range of airfoil angles of attack with flap deflections from 10 to 55 degrees and various leading edge configurations. Steady suction control was more effective at eliminating the large separation bubble created by the model, requiring a pressure ratio between the applied force and inviscid force of approximately unity, whereas blowing required a two to one ratio. Pulsed suction was superior and enhanced by the operating frequency or duty cycle. Separation control (SC) was modified by the presence of circulation control (CC). Steady-blowing SC near the leading edge reduced the effect of blown-flap CC, whereas steady-suction SC increased the performance gain.

  15. Elizabethkingia anophelis: Molecular Manipulation and Interactions with Mosquito Hosts

    PubMed Central

    Bagdasarian, Michael; Walker, Edward D.

    2015-01-01

    Flavobacteria (members of the family Flavobacteriaceae) dominate the bacterial community in the Anopheles mosquito midgut. One such commensal, Elizabethkingia anophelis, is closely associated with Anopheles mosquitoes through transstadial persistence (i.e., from one life stage to the next); these and other properties favor its development for paratransgenic applications in control of malaria parasite transmission. However, the physiological requirements of E. anophelis have not been investigated, nor has its capacity to perpetuate despite digestion pressure in the gut been quantified. To this end, we first developed techniques for genetic manipulation of E. anophelis, including selectable markers, reporter systems (green fluorescent protein [GFP] and NanoLuc), and transposons that function in E. anophelis. A flavobacterial expression system based on the promoter PompA was integrated into the E. anophelis chromosome and showed strong promoter activity to drive GFP and NanoLuc reporter production. Introduced, GFP-tagged E. anophelis associated with mosquitoes at successive developmental stages and propagated in Anopheles gambiae and Anopheles stephensi but not in Aedes triseriatus mosquitoes. Feeding NanoLuc-tagged cells to A. gambiae and A. stephensi in the larval stage led to infection rates of 71% and 82%, respectively. In contrast, a very low infection rate (3%) was detected in Aedes triseriatus mosquitoes under the same conditions. Of the initial E. anophelis cells provided to larvae, 23%, 71%, and 85% were digested in A. stephensi, A. gambiae, and Aedes triseriatus, respectively, demonstrating that E. anophelis adapted to various mosquito midgut environments differently. Bacterial cell growth increased up to 3-fold when arginine was supplemented in the defined medium. Furthermore, the number of NanoLuc-tagged cells in A. stephensi significantly increased when arginine was added to a sugar diet, showing it to be an important amino acid for E. anophelis. Animal

  16. Elizabethkingia anophelis: molecular manipulation and interactions with mosquito hosts.

    PubMed

    Chen, Shicheng; Bagdasarian, Michael; Walker, Edward D

    2015-03-01

    Flavobacteria (members of the family Flavobacteriaceae) dominate the bacterial community in the Anopheles mosquito midgut. One such commensal, Elizabethkingia anophelis, is closely associated with Anopheles mosquitoes through transstadial persistence (i.e., from one life stage to the next); these and other properties favor its development for paratransgenic applications in control of malaria parasite transmission. However, the physiological requirements of E. anophelis have not been investigated, nor has its capacity to perpetuate despite digestion pressure in the gut been quantified. To this end, we first developed techniques for genetic manipulation of E. anophelis, including selectable markers, reporter systems (green fluorescent protein [GFP] and NanoLuc), and transposons that function in E. anophelis. A flavobacterial expression system based on the promoter PompA was integrated into the E. anophelis chromosome and showed strong promoter activity to drive GFP and NanoLuc reporter production. Introduced, GFP-tagged E. anophelis associated with mosquitoes at successive developmental stages and propagated in Anopheles gambiae and Anopheles stephensi but not in Aedes triseriatus mosquitoes. Feeding NanoLuc-tagged cells to A. gambiae and A. stephensi in the larval stage led to infection rates of 71% and 82%, respectively. In contrast, a very low infection rate (3%) was detected in Aedes triseriatus mosquitoes under the same conditions. Of the initial E. anophelis cells provided to larvae, 23%, 71%, and 85% were digested in A. stephensi, A. gambiae, and Aedes triseriatus, respectively, demonstrating that E. anophelis adapted to various mosquito midgut environments differently. Bacterial cell growth increased up to 3-fold when arginine was supplemented in the defined medium. Furthermore, the number of NanoLuc-tagged cells in A. stephensi significantly increased when arginine was added to a sugar diet, showing it to be an important amino acid for E. anophelis. Animal

  17. Simulation of man-machine interaction on shuttle payload manipulator

    NASA Technical Reports Server (NTRS)

    Hookway, R. O.; Jackson, R. S.

    1975-01-01

    The main objective of this simulation was to evaluate the feasibility of a simplified control system for a remote manipulator for space shuttle payloads. The motion commanded by the operator through the control system to the six degree of freedom manipulator approximates that of a backhoe. Compatibility of low arm damping, heavy payloads, small clearances in the shuttle cargo bay and stringent mission timelines were evaluated. The effects of various devices to enhance visual cues were evaluated. Phase I of the simulation was capture of a payload flying free in space relative to the shuttle. Phase II was simulation of cargo stowage into a mockup of the space shuttle cargo bay. A shuttle remote manipulator control station mockup including TV monitors and hand controllers is used in the simulation. Results evaluating various parameters of the control system and the task, including arm flexibility, are presented.

  18. Carbohydrate–Aromatic Interactions in Proteins

    PubMed Central

    2015-01-01

    Protein–carbohydrate interactions play pivotal roles in health and disease. However, defining and manipulating these interactions has been hindered by an incomplete understanding of the underlying fundamental forces. To elucidate common and discriminating features in carbohydrate recognition, we have analyzed quantitatively X-ray crystal structures of proteins with noncovalently bound carbohydrates. Within the carbohydrate-binding pockets, aliphatic hydrophobic residues are disfavored, whereas aromatic side chains are enriched. The greatest preference is for tryptophan with an increased prevalence of 9-fold. Variations in the spatial orientation of amino acids around different monosaccharides indicate specific carbohydrate C–H bonds interact preferentially with aromatic residues. These preferences are consistent with the electronic properties of both the carbohydrate C–H bonds and the aromatic residues. Those carbohydrates that present patches of electropositive saccharide C–H bonds engage more often in CH−π interactions involving electron-rich aromatic partners. These electronic effects are also manifested when carbohydrate–aromatic interactions are monitored in solution: NMR analysis indicates that indole favorably binds to electron-poor C–H bonds of model carbohydrates, and a clear linear free energy relationships with substituted indoles supports the importance of complementary electronic effects in driving protein–carbohydrate interactions. Together, our data indicate that electrostatic and electronic complementarity between carbohydrates and aromatic residues play key roles in driving protein–carbohydrate complexation. Moreover, these weak noncovalent interactions influence which saccharide residues bind to proteins, and how they are positioned within carbohydrate-binding sites. PMID:26561965

  19. Cooperation and conflict in host manipulation: interactions among macro-parasites and micro-organisms

    PubMed Central

    Cézilly, Frank; Perrot-Minnot, Marie-Jeanne; Rigaud, Thierry

    2014-01-01

    Several parasite species are known to manipulate the phenotype of their hosts in ways that enhance their own transmission. Co-occurrence of manipulative parasites, belonging to the same species or to more than one species, in a single host has been regularly observed. Little is known, however, on interactions between co-occurring manipulative parasites with same or different transmission routes. Several models addressing this problem have provided predictions on how cooperation and conflict between parasites could emerge from multiple infections. Here, we review the empirical evidence in favor of the existence of synergistic or antagonistic interactions between co-occurring parasites, and highlight the neglected role of micro-organisms. We particularly discuss the actual importance of selective forces shaping the evolution of interactions between manipulative parasites in relation to parasite prevalence in natural populations, efficiency in manipulation, and type of transmission (i.e., horizontal versus vertical), and we emphasize the potential for future research. PMID:24966851

  20. PINT: Protein-protein Interactions Thermodynamic Database.

    PubMed

    Kumar, M D Shaji; Gromiha, M Michael

    2006-01-01

    The first release of Protein-protein Interactions Thermodynamic Database (PINT) contains >1500 data of several thermodynamic parameters along with sequence and structural information, experimental conditions and literature information. Each entry contains numerical data for the free energy change, dissociation constant, association constant, enthalpy change, heat capacity change and so on of the interacting proteins upon binding, which are important for understanding the mechanism of protein-protein interactions. PINT also includes the name and source of the proteins involved in binding, their Protein Information Resource, SWISS-PROT and Protein Data Bank (PDB) codes, secondary structure and solvent accessibility of residues at mutant positions, measuring methods, experimental conditions, such as buffers, ions and additives, and literature information. A WWW interface facilitates users to search data based on various conditions, feasibility to select the terms for output and different sorting options. Further, PINT is cross-linked with other related databases, PIR, SWISS-PROT, PDB and NCBI PUBMED literature database. The database is freely available at http://www.bioinfodatabase.com/pint/index.html. PMID:16381844

  1. Biochemical Approaches for Discovering Protein-Protein Interactions

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Protein-protein interactions or protein complexes are indigenous to nearly all cellular processes, ranging from metabolism to structure. Elucidating both individual protein associations and complex protein interaction networks, while challenging, is an essential goal of functional genomics. For ex...

  2. Understanding the Molecular Manipulation of DCAF1 by the Lentiviral Accessory Proteins Vpr and Vpx

    PubMed Central

    Chumley, Jeffrey; Ward, Jeffrey; Barker, Edward; Planelles, Vicente

    2014-01-01

    Vpr and Vpx are primate lentivirus proteins that manipulate the cellular CRL4 ubiquitin ligase complex. While Vpr is common to all primate lentiviruses, Vpx is only encoded by HIV-2 and a limited range of SIVs. Although Vpr and Vpx share a high degree of homology they are known to induce markedly different effects in host cell biology through the recruitment of different substrates to CRL4. Here we explore the interaction of HIV-1 Vpr and SIVmac Vpx with the CRL4 substrate receptor DCAF1. Through mutational analysis of DCAF1 we demonstrate that although Vpr and Vpx share a highly similar DCAF1-binding motif, they interact with a different set of residues in DCAF1. In addition, we show that Vpx recruits SAMHD1 through a protein-protein interface that includes interactions of SAMHD1 with both Vpx and DCAF1, as was first suggested in crystallography data by Schwefel et al. (Nature 505:234, 2014). PMID:25499532

  3. Effects of manipulation of the caspase system on myofibrillar protein degradation in vitro.

    PubMed

    Kemp, C M; Wheeler, T L

    2011-10-01

    Apoptosis via the intrinsic caspase 9 pathway can be induced by oxidative stressors hydrogen peroxide (H₂O₂) and N-(4 hydroxyphenol) rentinamide (fenretinide), a synthetic retinoid. Accelerated muscle atrophy and proteolysis in muscle-wasting conditions have been linked to oxidative stress and activated protease systems. Therefore, the hypothesis of this study was that proteolysis of myofibrillar proteins could be manipulated through the induction or inhibition of the caspase system. After slaughter, LM and supraspinatus muscles from callipyge (n = 5) and normal (n = 3) lambs were excised, finely diced, and incubated with treatment buffers containing oxidative stressors fenretinide or H₂O₂, recombinant caspase 3, caspase-specific inhibitor N-acetyl-Asp-Glu-Val-Asp-CHO (DEVD), or control solution. Muscle samples were incubated for 1, 2, 7, and 21 d at 4°C. Activation of the initiator caspase, caspase 9, and myofibrillar protein degradation was determined by SDS-PAGE and Western blotting. Results showed that fenretinide, H₂O₂, and recombinant caspase 3 increased (P < 0.05) proteolysis of myofibril proteins, whereas DEVD inhibited degradation (P < 0.05). Proteolysis of myofibrillar proteins increased with incubation time (P < 0.0001), and incubation time × treatment interactions (P < 0.05) indicated that the treatment effects did not all occur at the same rate. This study has shown that manipulation of the caspase system through induction or inhibition of activity can affect degradation of myofibrillar proteins, providing further evidence that the caspase system could be involved in postmortem proteolysis and tenderization. However, these stimulated changes were not sufficient to overcome the lack of proteolysis that is characteristic of muscle from callipyge lambs. PMID:21622882

  4. Electrical manipulation of spins in a nanowire with Rashba interaction

    NASA Astrophysics Data System (ADS)

    Sakr, M. R.

    2016-07-01

    We investigate the influence of external electric fields on the spins of a ballistic nanowire in terms of variations of the Rashba parameter and modification of the confinement potential. For a weak Rashba effect, the spins along the confinement direction in a given subband nearly assume full quantization. In the presence of a perpendicular magnetic field, the state of quantization can be manipulated using a transverse electric. This process requires modifications in the spin textures. If an in-plane magnetic field is applied, spins suffer rigid displacement to one edge of the wire and their expectation value becomes independent of the transverse electric field.

  5. Domains mediate protein-protein interactions and nucleate protein assemblies.

    PubMed

    Costa, S; Cesareni, G

    2008-01-01

    Cell physiology is governed by an intricate mesh of physical and functional links among proteins, nucleic acids and other metabolites. The recent information flood coming from large-scale genomic and proteomic approaches allows us to foresee the possibility of compiling an exhaustive list of the molecules present within a cell, enriched with quantitative information on concentration and cellular localization. Moreover, several high-throughput experimental and computational techniques have been devised to map all the protein interactions occurring in a living cell. So far, such maps have been drawn as graphs where nodes represent proteins and edges represent interactions. However, this representation does not take into account the intrinsically modular nature of proteins and thus fails in providing an effective description of the determinants of binding. Since proteins are composed of domains that often confer on proteins their binding capabilities, a more informative description of the interaction network would detail, for each pair of interacting proteins in the network, which domains mediate the binding. Understanding how protein domains combine to mediate protein interactions would allow one to add important features to the protein interaction network, making it possible to discriminate between simultaneously occurring and mutually exclusive interactions. This objective can be achieved by experimentally characterizing domain recognition specificity or by analyzing the frequency of co-occurring domains in proteins that do interact. Such approaches allow gaining insights on the topology of complexes with unknown three-dimensional structure, thus opening the prospect of adopting a more rational strategy in developing drugs designed to selectively target specific protein interactions. PMID:18491061

  6. Bacteriophage Protein–Protein Interactions

    PubMed Central

    Häuser, Roman; Blasche, Sonja; Dokland, Terje; Haggård-Ljungquist, Elisabeth; von Brunn, Albrecht; Salas, Margarita; Casjens, Sherwood; Molineux, Ian

    2012-01-01

    Bacteriophages T7, λ, P22, and P2/P4 (from Escherichia coli), as well as ϕ29 (from Bacillus subtilis), are among the best-studied bacterial viruses. This chapter summarizes published protein interaction data of intraviral protein interactions, as well as known phage–host protein interactions of these phages retrieved from the literature. We also review the published results of comprehensive protein interaction analyses of Pneumococcus phages Dp-1 and Cp-1, as well as coliphages λ and T7. For example, the ≈55 proteins encoded by the T7 genome are connected by ≈43 interactions with another ≈15 between the phage and its host. The chapter compiles published interactions for the well-studied phages λ (33 intra-phage/22 phage-host), P22 (38/9), P2/P4 (14/3), and ϕ29 (20/2). We discuss whether different interaction patterns reflect different phage lifestyles or whether they may be artifacts of sampling. Phages that infect the same host can interact with different host target proteins, as exemplified by E. coli phage λ and T7. Despite decades of intensive investigation, only a fraction of these phage interactomes are known. Technical limitations and a lack of depth in many studies explain the gaps in our knowledge. Strategies to complete current interactome maps are described. Although limited space precludes detailed overviews of phage molecular biology, this compilation will allow future studies to put interaction data into the context of phage biology. PMID:22748812

  7. Manipulation of Plant Host Susceptibility: An Emerging Role for Viral Movement Proteins?

    PubMed Central

    Amari, Khalid; Vazquez, Franck; Heinlein, Manfred

    2012-01-01

    Viruses encode viral suppressors of RNA silencing (VSRs) to counteract RNA silencing, a major antiviral defense response in plants. Recent studies indicate a role of virus-derived siRNAs in manipulating the expression of specific host genes and that certain plant viral movement proteins (MPs) can act as viral enhancers of RNA silencing (VERs) by stimulating the spread of silencing between cells. This suggests that viruses have evolved complex responses capable to efficiently hijack the host RNA silencing machinery to their own advantage. We draw here a dynamic model of the interaction of plant viruses with the silencing machinery during invasion of the host. The model proposes that cells at the spreading front of infection, where infection starts from zero and the VSR levels are supposedly low, represent potential sites for viral manipulation of host gene expression by using virus- and host-derived small RNAs. Viral MPs may facilitate the spread of silencing to produce a wave of small RNA-mediated gene expression changes ahead of the infection to increase host susceptibility. When experimentally ascertained, this hypothetical model will call for re-defining viral movement and the function of viral MPs. PMID:22639637

  8. Protein-protein interactions as drug targets.

    PubMed

    Skwarczynska, Malgorzata; Ottmann, Christian

    2015-10-01

    Modulation of protein-protein interactions (PPIs) is becoming increasingly important in drug discovery and chemical biology. While a few years ago this 'target class' was deemed to be largely undruggable an impressing number of publications and success stories now show that targeting PPIs with small, drug-like molecules indeed is a feasible approach. Here, we summarize the current state of small-molecule inhibition and stabilization of PPIs and review the active molecules from a structural and medicinal chemistry angle, especially focusing on the key examples of iNOS, LFA-1 and 14-3-3. PMID:26510391

  9. Recognition of haptic interaction patterns in dyadic joint object manipulation.

    PubMed

    Madan, Cigil Ece; Kucukyilmaz, Ayse; Sezgin, Tevfik Metin; Basdogan, Cagatay

    2015-01-01

    The development of robots that can physically cooperate with humans has attained interest in the last decades. Obviously, this effort requires a deep understanding of the intrinsic properties of interaction. Up to now, many researchers have focused on inferring human intents in terms of intermediate or terminal goals in physical tasks. On the other hand, working side by side with people, an autonomous robot additionally needs to come up with in-depth information about underlying haptic interaction patterns that are typically encountered during human-human cooperation. However, to our knowledge, no study has yet focused on characterizing such detailed information. In this sense, this work is pioneering as an effort to gain deeper understanding of interaction patterns involving two or more humans in a physical task. We present a labeled human-human-interaction dataset, which captures the interaction of two humans, who collaboratively transport an object in an haptics-enabled virtual environment. In the light of information gained by studying this dataset, we propose that the actions of cooperating partners can be examined under three interaction types: In any cooperative task, the interacting humans either 1) work in harmony, 2) cope with conflicts, or 3) remain passive during interaction. In line with this conception, we present a taxonomy of human interaction patterns; then propose five different feature sets, comprising force-, velocity-and power-related information, for the classification of these patterns. Our evaluation shows that using a multi-class support vector machine (SVM) classifier, we can accomplish a correct classification rate of 86 percent for the identification of interaction patterns, an accuracy obtained by fusing a selected set of most informative features by Minimum Redundancy Maximum Relevance (mRMR) feature selection method. PMID:25532210

  10. Manipulating Cognitive Complexity across Task Types and Its Impact on Learners' Interaction during Oral Performance

    ERIC Educational Resources Information Center

    Gilabert, Roger; Baron, Julia; Llanes, Angels

    2009-01-01

    The goal of this study is to investigate the impact of manipulating the cognitive complexity of three different types of oral tasks on interaction. The study first considers the concepts of task complexity and interaction and then examines the specific studies that have looked at the effects of increasing task complexity on conversational…

  11. Split-Protein Systems: Beyond Binary Protein-Protein Interactions

    PubMed Central

    Shekhawat, Sujan S.; Ghosh, Indraneel

    2011-01-01

    It has been estimated that 650,000 protein-protein interactions exist in the human interactome [1], a subset of all possible macromolecular partnerships that dictate life. Thus there is a continued need for the development of sensitive and user-friendly methods for cataloguing biomacromolecules in complex environments and for detecting their interactions, modifications, and cellular location. Such methods also allow for establishing differences in the interactome between a normal and diseased cellular state and for quantifying the outcome of therapeutic intervention. A promising approach for deconvoluting the role of macromolecular partnerships is split-protein reassembly, also called protein fragment complementation. This approach relies on the appropriate fragmentation of protein reporters, such as the green fluorescent protein or firefly luciferase, which when attached to possible interacting partners can reassemble and regain function, thereby confirming the partnership. Split-protein methods have been effectively utilized for detecting protein-protein interactions in cell-free systems, E. coli, yeast, mammalian cells, plants, and live animals. Herein, we present recent advances in engineering split-protein systems that allow for the rapid detection of ternary protein complexes, small molecule inhibitors, as well as a variety of macromolecules including nucleic acids, poly(ADP) ribose, and iron sulfur clusters. We also present advances that combine split-protein systems with chemical inducers of dimerization strategies that allow for regulating the activity of orthogonal split-proteases as well as aid in identifying enzyme inhibitors. Finally, we discuss autoinhibition strategies leading to turn-on sensors as well as future directions in split-protein methodology including possible therapeutic approaches. PMID:22070901

  12. Split-protein systems: beyond binary protein-protein interactions.

    PubMed

    Shekhawat, Sujan S; Ghosh, Indraneel

    2011-12-01

    It has been estimated that 650,000 protein-protein interactions exist in the human interactome (Stumpf et al., 2008), a subset of all possible macromolecular partnerships that dictate life. Thus there is a continued need for the development of sensitive and user-friendly methods for cataloguing biomacromolecules in complex environments and for detecting their interactions, modifications, and cellular location. Such methods also allow for establishing differences in the interactome between a normal and diseased cellular state and for quantifying the outcome of therapeutic intervention. A promising approach for deconvoluting the role of macromolecular partnerships is split-protein reassembly, also called protein fragment complementation. This approach relies on the appropriate fragmentation of protein reporters, such as the green fluorescent protein or firefly luciferase, which when attached to possible interacting partners can reassemble and regain function, thereby confirming the partnership. Split-protein methods have been effectively utilized for detecting protein-protein interactions in cell-free systems, Escherichia coli, yeast, mammalian cells, plants, and live animals. Herein, we present recent advances in engineering split-protein systems that allow for the rapid detection of ternary protein complexes, small molecule inhibitors, as well as a variety of macromolecules including nucleic acids, poly(ADP) ribose, and iron sulfur clusters. We also present advances that combine split-protein systems with chemical inducers of dimerization strategies that allow for regulating the activity of orthogonal split-proteases as well as aid in identifying enzyme inhibitors. Finally, we discuss autoinhibition strategies leading to turn-on sensors as well as future directions in split-protein methodology including possible therapeutic approaches. PMID:22070901

  13. Energy design for protein-protein interactions

    PubMed Central

    Ravikant, D. V. S.; Elber, Ron

    2011-01-01

    Proteins bind to other proteins efficiently and specifically to carry on many cell functions such as signaling, activation, transport, enzymatic reactions, and more. To determine the geometry and strength of binding of a protein pair, an energy function is required. An algorithm to design an optimal energy function, based on empirical data of protein complexes, is proposed and applied. Emphasis is made on negative design in which incorrect geometries are presented to the algorithm that learns to avoid them. For the docking problem the search for plausible geometries can be performed exhaustively. The possible geometries of the complex are generated on a grid with the help of a fast Fourier transform algorithm. A novel formulation of negative design makes it possible to investigate iteratively hundreds of millions of negative examples while monotonically improving the quality of the potential. Experimental structures for 640 protein complexes are used to generate positive and negative examples for learning parameters. The algorithm designed in this work finds the correct binding structure as the lowest energy minimum in 318 cases of the 640 examples. Further benchmarks on independent sets confirm the significant capacity of the scoring function to recognize correct modes of interactions. PMID:21842951

  14. Energy design for protein-protein interactions

    NASA Astrophysics Data System (ADS)

    Ravikant, D. V. S.; Elber, Ron

    2011-08-01

    Proteins bind to other proteins efficiently and specifically to carry on many cell functions such as signaling, activation, transport, enzymatic reactions, and more. To determine the geometry and strength of binding of a protein pair, an energy function is required. An algorithm to design an optimal energy function, based on empirical data of protein complexes, is proposed and applied. Emphasis is made on negative design in which incorrect geometries are presented to the algorithm that learns to avoid them. For the docking problem the search for plausible geometries can be performed exhaustively. The possible geometries of the complex are generated on a grid with the help of a fast Fourier transform algorithm. A novel formulation of negative design makes it possible to investigate iteratively hundreds of millions of negative examples while monotonically improving the quality of the potential. Experimental structures for 640 protein complexes are used to generate positive and negative examples for learning parameters. The algorithm designed in this work finds the correct binding structure as the lowest energy minimum in 318 cases of the 640 examples. Further benchmarks on independent sets confirm the significant capacity of the scoring function to recognize correct modes of interactions.

  15. Single-Molecule Manipulation Studies of a Mechanically Activated Protein

    NASA Astrophysics Data System (ADS)

    Botello, Eric; Harris, Nolan; Choi, Huiwan; Bergeron, Angela; Dong, Jing-Fei; Kiang, Ching-Hwa

    2009-10-01

    Plasma von Willebrand factor (pVWF) is the largest multimeric adhesion ligand found in human blood and must be adhesively activated by exposure to shear stress, like at sites of vascular injury, to initiate blood clotting. Sheared pVWF (sVWF) will undergo a conformational change from a loose tangled coil to elongated strings forming adhesive fibers by binding with other sVWF. VWF's adhesion activity is also related to its length, with the ultra-large form of VWF (ULVWF) being hyper-actively adhesive without exposure to shear stress; it has also been shown to spontaneously form fibers. We used single molecule manipulation techniques with the AFM to stretch pVWF, sVWF and ULVWF and monitor the forces as a function of molecular extension. We showed a similar increase in resistance to unfolding for sVWF and ULVWF when compared to pVWF. This mechanical resistance to forced unfolding is reduced when other molecules known to disrupt their fibril formation are present. Our results show that sVWF and ULVWF domains unfold at higher forces than pVWF, which is consistent with the hypothesis that shear stress induces lateral association that alters adhesion activity of pVWF.

  16. Interactions of Pathological Hallmark Proteins

    PubMed Central

    Oláh, Judit; Vincze, Orsolya; Virók, Dezső; Simon, Dóra; Bozsó, Zsolt; Tőkési, Natália; Horváth, István; Hlavanda, Emma; Kovács, János; Magyar, Anna; Szűcs, Mária; Orosz, Ferenc; Penke, Botond; Ovádi, Judit

    2011-01-01

    The disordered tubulin polymerization promoting protein (TPPP/p25) was found to be co-enriched in neuronal and glial inclusions with α-synuclein in Parkinson disease and multiple system atrophy, respectively; however, co-occurrence of α-synuclein with β-amyloid (Aβ) in human brain inclusions has been recently reported, suggesting the existence of mixed type pathologies that could result in obstacles in the correct diagnosis and treatment. Here we identified TPPP/p25 as an interacting partner of the soluble Aβ oligomers as major risk factors for Alzheimer disease using ProtoArray human protein microarray. The interactions of oligomeric Aβ with proteins involved in the etiology of neurological disorders were characterized by ELISA, surface plasmon resonance, pelleting experiments, and tubulin polymerization assay. We showed that the Aβ42 tightly bound to TPPP/p25 (Kd = 85 nm) and caused aberrant protein aggregation by inhibiting the physiologically relevant TPPP/p25-derived microtubule assembly. The pair-wise interactions of Aβ42, α-synuclein, and tubulin were found to be relatively weak; however, these three components formed soluble ternary complex exclusively in the absence of TPPP/p25. The aggregation-facilitating activity of TPPP/p25 and its interaction with Aβ was monitored by electron microscopy with purified proteins by pelleting experiments with cell-free extracts as well as by confocal microscopy with CHO cells expressing TPPP/p25 or amyloid. The finding that the interaction of TPPP/p25 with Aβ can produce pathological-like aggregates is tightly coupled with unusual pathology of the Alzheimer disease revealed previously; that is, partial co-localization of Aβ and TPPP/p25 in the case of diffuse Lewy body disease with Alzheimer disease. PMID:21832049

  17. Protein- protein interaction detection system using fluorescent protein microdomains

    DOEpatents

    Waldo, Geoffrey S.; Cabantous, Stephanie

    2010-02-23

    The invention provides a protein labeling and interaction detection system based on engineered fragments of fluorescent and chromophoric proteins that require fused interacting polypeptides to drive the association of the fragments, and further are soluble and stable, and do not change the solubility of polypeptides to which they are fused. In one embodiment, a test protein X is fused to a sixteen amino acid fragment of GFP (.beta.-strand 10, amino acids 198-214), engineered to not perturb fusion protein solubility. A second test protein Y is fused to a sixteen amino acid fragment of GFP (.beta.-strand 11, amino acids 215-230), engineered to not perturb fusion protein solubility. When X and Y interact, they bring the GFP strands into proximity, and are detected by complementation with a third GFP fragment consisting of GFP amino acids 1-198 (strands 1-9). When GFP strands 10 and 11 are held together by interaction of protein X and Y, they spontaneous association with GFP strands 1-9, resulting in structural complementation, folding, and concomitant GFP fluorescence.

  18. Manipulating Cofactor Binding Thermodynamics in an Artificial Oxygen Transport Protein

    PubMed Central

    Zhang, Lei; Ross Anderson, J. L.; Ahmed, Ismail; Norman, Jessica A.; Negron, Christopher; Mutter, Andrew C.; Dutton, P. Leslie; Koder, Ronald L.

    2013-01-01

    We report the mutational analysis of an artificial oxygen transport protein, HP-7, which operates via a mechanism akin to human neuroglobin and cytoglobin. This protein destabilizes one of two heme-ligating histidine residues by coupling histidine side chain ligation with the burial of three charged glutamate residues on the same helix. Replacement of these glutamate residues with alanine, which is uncharged, increases the affinity of the distal histidine ligand by a factor of thirteen. Paradoxically, it also decreases heme binding affinity by a factor of five in the reduced state and sixty in the oxidized state. Application of a three-state binding model, in which an initial pentacoordinate binding event is followed by a protein conformational change to hexacoordinate, provides insight into the mechanism of this seemingly counterintuitive result: the initial pentacoordinate encounter complex is significantly destabilized by the loss of the glutamate side chains, and the increased affinity for the distal histidine only partially compensates. These results point to the importance of considering each oxidation and conformational state in the design of functional artificial proteins. PMID:22004125

  19. Direct Probing of Protein-Protein Interactions

    SciTech Connect

    Noy, A; Sulchek, T A; Friddle, R W

    2005-03-10

    This project aimed to establish feasibility of using experimental techniques based on direct measurements of interaction forces on the single molecule scale to characterize equilibrium interaction potentials between individual biological molecules. Such capability will impact several research areas, ranging from rapid interaction screening capabilities to providing verifiable inputs for computational models. It should be one of the enabling technologies for modern proteomics research. This study used a combination of Monte-Carlo simulations, theoretical considerations, and direct experimental measurements to investigate two model systems that represented typical experimental situations: force-induced melting of DNA rigidly attached to the tip, and force-induced unbinding of a protein-antibody pair connected to flexible tethers. Our results establish that for both systems researchers can use force spectroscopy measurements to extract reliable information about equilibrium interaction potentials. However, the approaches necessary to extract these potentials in each case--Jarzynski reconstruction and Dynamic Force Spectroscopy--are very different. We also show how the thermodynamics and kinetics of unbinding process dictates the choice between in each case.

  20. Experimental manipulation of floral scent bouquets restructures flower-visitor interactions in the field.

    PubMed

    Larue, Anne-Amélie C; Raguso, Robert A; Junker, Robert R

    2016-03-01

    A common structural feature of natural communities is the non-random distribution of pairwise interactions between organisms of different trophic levels. For plant-animal interactions, it is predicted that both stochastic processes and functional plant traits that facilitate or prevent interactions are responsible for these patterns. However, unbiased manipulative field experiments that rigorously test the effects of individual traits on community structure are lacking. We address this gap by manipulating floral scent bouquets in the field. Manipulation of floral scent bouquets led to quantitative as well as qualitative restructuring of flower-visitor networks, making them more generalized. Olfactometer trials confirmed both positive and negative responses to scent bouquets. Our results clearly show that the distribution of insect visitors to the two abundant study plant species reflects the insects' species-specific preferences for floral scents, rather than for visual or morphological floral traits. Thus, floral scents may be of major importance in partitioning flower-visitor interactions. Integrating experimental manipulations of plant traits with field observations of interaction patterns thus represents a promising approach for revealing the processes that structure species assemblages in natural communities. PMID:26428739

  1. The impact of interactive manipulation on the recognition of objects

    NASA Astrophysics Data System (ADS)

    Meijer, Frank; van den Broek, Egon L.; Schouten, Theo

    2008-02-01

    A new application for VR has emerged: product development, in which several stakeholders (from engineers to end users) use the same VR for development and communicate purposes. Various characteristics among these stakeholders vary considerably, which imposes potential constraints to the VR. The current paper discusses the influence of three types of exploration of objects (i.e., none, passive, active) on one of these characteristics: the ability to form mental representations or visuo-spatial ability (VSA). Through an experiment we found that all users benefit from exploring objects. Moreover, people with low VSA (e.g., end users) benefit from an interactive exploration of objects opposed to people with a medium or high VSA (e.g. engineers), who are not sensitive for the type of exploration. Hence, for VR environments in which multiple stakeholders participate (e.g. for product development), differences among their cognitive abilities (e.g., VSA) have to be taken into account to enable an efficient usage of VR.

  2. Hydrodynamic interactions in protein folding

    NASA Astrophysics Data System (ADS)

    Cieplak, Marek; Niewieczerzał, Szymon

    2009-03-01

    We incorporate hydrodynamic interactions (HIs) in a coarse-grained and structure-based model of proteins by employing the Rotne-Prager hydrodynamic tensor. We study several small proteins and demonstrate that HIs facilitate folding. We also study HIV-1 protease and show that HIs make the flap closing dynamics faster. The HIs are found to affect time correlation functions in the vicinity of the native state even though they have no impact on same time characteristics of the structure fluctuations around the native state.

  3. Hydrodynamic interactions in protein folding.

    PubMed

    Cieplak, Marek; Niewieczerzał, Szymon

    2009-03-28

    We incorporate hydrodynamic interactions (HIs) in a coarse-grained and structure-based model of proteins by employing the Rotne-Prager hydrodynamic tensor. We study several small proteins and demonstrate that HIs facilitate folding. We also study HIV-1 protease and show that HIs make the flap closing dynamics faster. The HIs are found to affect time correlation functions in the vicinity of the native state even though they have no impact on same time characteristics of the structure fluctuations around the native state. PMID:19334888

  4. Examination and Manipulation of Protein Surface Charge in Solution with Electrospray Ionization Mass Spectrometry

    ERIC Educational Resources Information Center

    Gross, Deborah S.; Van Ryswyk, Hal

    2014-01-01

    Electrospray ionization mass spectrometry (ESI-MS) is a powerful tool for examining the charge of proteins in solution. The charge can be manipulated through choice of solvent and pH. Furthermore, solution-accessible, protonated lysine side chains can be specifically tagged with 18-crown-6 ether to form noncovalent adducts. Chemical derivatization…

  5. Quantifying molecule-surface interactions using AFM-based single-molecule manipulation

    NASA Astrophysics Data System (ADS)

    Tautz, F. S.; Wagner, C.; Temirov, R.; Fournier, N.; Green, M.; Esat, T.; Leinen, P.; Groetsch, A.; Ruiz, V. G.; Tkatchenko, A.; Li, C.; Muellen, K.; Rohlfing, M.

    2015-03-01

    Scanning probe microscopy plays an important role in the investigation of molecular adsorption. Promising, is the possibility to probe the molecule-surface interaction while tuning its strength through AFM tip-induced single-molecule manipulation. Here, we outline a strategy to achieve quantitative understanding of such manipulation experiments. The example of qPlus sensor based PTCDA molecule lifting experiments is used to demonstrate how different aspects of the molecule-surface interaction, namely the short-range adsorption potential, the asymptotic van der Waals potential, local chemical bonds which are the source of the surface corrugation, and molecule-molecule interactions can be measured with SPM and interpreted by the help of force-field simulations.

  6. Survey on indirect optical manipulation of cells, nucleic acids, and motor proteins.

    PubMed

    Banerjee, Ashis Gopal; Chowdhury, Sagar; Losert, Wolfgang; Gupta, Satyandra K

    2011-05-01

    Optical tweezers have emerged as a promising technique for manipulating biological objects. Instead of direct laser exposure, more often than not, optically-trapped beads are attached to the ends or boundaries of the objects for translation, rotation, and stretching. This is referred to as indirect optical manipulation. In this paper, we utilize the concept of robotic gripping to explain the different experimental setups which are commonly used for indirect manipulation of cells, nucleic acids, and motor proteins. We also give an overview of the kind of biological insights provided by this technique. We conclude by highlighting the trends across the experimental studies, and discuss challenges and promising directions in this domain of active current research. PMID:21639562

  7. Protein-Inhibitor Interaction Studies Using NMR

    PubMed Central

    Ishima, Rieko

    2015-01-01

    Solution-state NMR has been widely applied to determine the three-dimensional structure, dynamics, and molecular interactions of proteins. The designs of experiments used in protein NMR differ from those used for small-molecule NMR, primarily because the information available prior to an experiment, such as molecular mass and knowledge of the primary structure, is unique for proteins compared to small molecules. In this review article, protein NMR for structural biology is introduced with comparisons to small-molecule NMR, such as descriptions of labeling strategies and the effects of molecular dynamics on relaxation. Next, applications for protein NMR are reviewed, especially practical aspects for protein-observed ligand-protein interaction studies. Overall, the following topics are described: (1) characteristics of protein NMR, (2) methods to detect protein-ligand interactions by NMR, and (3) practical aspects of carrying out protein-observed inhibitor-protein interaction studies. PMID:26361636

  8. Longitudinal phase space manipulation of an ultrashort electron beam via THz IFEL interaction

    SciTech Connect

    Moody, J. T.; Li, R. K.; Musumeci, P.; Scoby, C. M.; To, H.

    2012-12-21

    A scheme where a laser locked THz source is used to manipulate the longitudinal phase space of an ultrashort electron beam using an IFEL interaction is investigated. The efficiency of THz source based on the pulse front tilt optical rectification scheme is increased by cryogenic cooling to achieve sufficient THz power for compression and synchronization. Start-to-end simulations describing the evolution of the beam from the cathode to the compression point after the undulator are presented.

  9. Bioinformatic Prediction of WSSV-Host Protein-Protein Interaction

    PubMed Central

    Sun, Zheng; Xiang, Jianhai

    2014-01-01

    WSSV is one of the most dangerous pathogens in shrimp aquaculture. However, the molecular mechanism of how WSSV interacts with shrimp is still not very clear. In the present study, bioinformatic approaches were used to predict interactions between proteins from WSSV and shrimp. The genome data of WSSV (NC_003225.1) and the constructed transcriptome data of F. chinensis were used to screen potentially interacting proteins by searching in protein interaction databases, including STRING, Reactome, and DIP. Forty-four pairs of proteins were suggested to have interactions between WSSV and the shrimp. Gene ontology analysis revealed that 6 pairs of these interacting proteins were classified into “extracellular region” or “receptor complex” GO-terms. KEGG pathway analysis showed that they were involved in the “ECM-receptor interaction pathway.” In the 6 pairs of interacting proteins, an envelope protein called “collagen-like protein” (WSSV-CLP) encoded by an early virus gene “wsv001” in WSSV interacted with 6 deduced proteins from the shrimp, including three integrin alpha (ITGA), two integrin beta (ITGB), and one syndecan (SDC). Sequence analysis on WSSV-CLP, ITGA, ITGB, and SDC revealed that they possessed the sequence features for protein-protein interactions. This study might provide new insights into the interaction mechanisms between WSSV and shrimp. PMID:24982879

  10. Proteins interacting with cloning scars: a source of false positive protein-protein interactions

    PubMed Central

    Banks, Charles A. S.; Boanca, Gina; Lee, Zachary T.; Florens, Laurence; Washburn, Michael P.

    2015-01-01

    A common approach for exploring the interactome, the network of protein-protein interactions in cells, uses a commercially available ORF library to express affinity tagged bait proteins; these can be expressed in cells and endogenous cellular proteins that copurify with the bait can be identified as putative interacting proteins using mass spectrometry. Control experiments can be used to limit false-positive results, but in many cases, there are still a surprising number of prey proteins that appear to copurify specifically with the bait. Here, we have identified one source of false-positive interactions in such studies. We have found that a combination of: 1) the variable sequence of the C-terminus of the bait with 2) a C-terminal valine “cloning scar” present in a commercially available ORF library, can in some cases create a peptide motif that results in the aberrant co-purification of endogenous cellular proteins. Control experiments may not identify false positives resulting from such artificial motifs, as aberrant binding depends on sequences that vary from one bait to another. It is possible that such cryptic protein binding might occur in other systems using affinity tagged proteins; this study highlights the importance of conducting careful follow-up studies where novel protein-protein interactions are suspected. PMID:25704442

  11. Coevolution of gene expression among interacting proteins

    SciTech Connect

    Fraser, Hunter B.; Hirsh, Aaron E.; Wall, Dennis P.; Eisen,Michael B.

    2004-03-01

    Physically interacting proteins or parts of proteins are expected to evolve in a coordinated manner that preserves proper interactions. Such coevolution at the amino acid-sequence level is well documented and has been used to predict interacting proteins, domains, and amino acids. Interacting proteins are also often precisely coexpressed with one another, presumably to maintain proper stoichiometry among interacting components. Here, we show that the expression levels of physically interacting proteins coevolve. We estimate average expression levels of genes from four closely related fungi of the genus Saccharomyces using the codon adaptation index and show that expression levels of interacting proteins exhibit coordinated changes in these different species. We find that this coevolution of expression is a more powerful predictor of physical interaction than is coevolution of amino acid sequence. These results demonstrate previously uncharacterized coevolution of gene expression, adding a different dimension to the study of the coevolution of interacting proteins and underscoring the importance of maintaining coexpression of interacting proteins over evolutionary time. Our results also suggest that expression coevolution can be used for computational prediction of protein protein interactions.

  12. Protein-protein interactions in DNA mismatch repair.

    PubMed

    Friedhoff, Peter; Li, Pingping; Gotthardt, Julia

    2016-02-01

    The principal DNA mismatch repair proteins MutS and MutL are versatile enzymes that couple DNA mismatch or damage recognition to other cellular processes. Besides interaction with their DNA substrates this involves transient interactions with other proteins which is triggered by the DNA mismatch or damage and controlled by conformational changes. Both MutS and MutL proteins have ATPase activity, which adds another level to control their activity and interactions with DNA substrates and other proteins. Here we focus on the protein-protein interactions, protein interaction sites and the different levels of structural knowledge about the protein complexes formed with MutS and MutL during the mismatch repair reaction. PMID:26725162

  13. An application of the MPP to the interactive manipulation of stereo images of digital terrain models

    NASA Technical Reports Server (NTRS)

    Pol, Sanjay; Mcallister, David; Davis, Edward

    1987-01-01

    Massively Parallel Processor algorithms were developed for the interactive manipulation of flat shaded digital terrain models defined over grids. The emphasis is on real time manipulation of stereo images. Standard graphics transformations are applied to a 128 x 128 grid of elevations followed by shading and a perspective projection to produce the right eye image. The surface is then rendered using a simple painter's algorithm for hidden surface removal. The left eye image is produced by rotating the surface 6 degs about the viewer's y axis followed by a perspective projection and rendering of the image as described above. The left and right eye images are then presented on a graphics device using standard stereo technology. Performance evaluations and comparisons are presented.

  14. Transient protein-protein interactions visualized by solution NMR.

    PubMed

    Liu, Zhu; Gong, Zhou; Dong, Xu; Tang, Chun

    2016-01-01

    Proteins interact with each other to establish their identities in cell. The affinities for the interactions span more than ten orders of magnitude, and KD values in μM-mM regimen are considered transient and are important in cell signaling. Solution NMR including diamagnetic and paramagnetic techniques has enabled atomic-resolution depictions of transient protein-protein interactions. Diamagnetic NMR allows characterization of protein complexes with KD values up to several mM, whereas ultraweak and fleeting complexes can be modeled with the use of paramagnetic NMR especially paramagnetic relaxation enhancement (PRE). When tackling ever-larger protein complexes, PRE can be particularly useful in providing long-range intermolecular distance restraints. As NMR measurements are averaged over the ensemble of complex structures, structural information for dynamic protein-protein interactions besides the stereospecific one can often be extracted. Herein the protein interaction dynamics are exemplified by encounter complexes, alternative binding modes, and coupled binding/folding of intrinsically disordered proteins. Further integration of NMR with other biophysical techniques should allow better visualization of transient protein-protein interactions. In particular, single-molecule data may facilitate the interpretation of ensemble-averaged NMR data. Though same structures of proteins and protein complexes were found in cell as in diluted solution, we anticipate that the dynamics of transient protein protein-protein interactions be different, which awaits awaits exploration by NMR. This article is part of a Special Issue entitled: Physiological Enzymology and Protein Functions. This article is part of a Special Issue entitled: Physiological Enzymology and Protein Functions. PMID:25896389

  15. How do oncoprotein mutations rewire protein-protein interaction networks?

    PubMed

    Bowler, Emily H; Wang, Zhenghe; Ewing, Rob M

    2015-01-01

    The acquisition of mutations that activate oncogenes or inactivate tumor suppressors is a primary feature of most cancers. Mutations that directly alter protein sequence and structure drive the development of tumors through aberrant expression and modification of proteins, in many cases directly impacting components of signal transduction pathways and cellular architecture. Cancer-associated mutations may have direct or indirect effects on proteins and their interactions and while the effects of mutations on signaling pathways have been widely studied, how mutations alter underlying protein-protein interaction networks is much less well understood. Systematic mapping of oncoprotein protein interactions using proteomics techniques as well as computational network analyses is revealing how oncoprotein mutations perturb protein-protein interaction networks and drive the cancer phenotype. PMID:26325016

  16. A Gateway-Based System for Fast Evaluation of Protein-Protein Interactions in Bacteria

    PubMed Central

    Wille, Thorsten; Barlag, Britta; Jakovljevic, Vladimir; Hensel, Michael; Sourjik, Victor; Gerlach, Roman G.

    2015-01-01

    Protein-protein interactions are important layers of regulation in all kingdoms of life. Identification and characterization of these interactions is one challenging task of the post-genomic era and crucial for understanding of molecular processes within a cell. Several methods have been successfully employed during the past decades to identify protein-protein interactions in bacteria, but most of them include tedious and time-consuming manipulations of DNA. In contrast, the MultiSite Gateway system is a fast tool for transfer of multiple DNA fragments between plasmids enabling simultaneous and site directed cloning of up to four fragments into one construct. Here we developed a new set of Gateway vectors including custom made entry vectors and modular Destination vectors for studying protein-protein interactions via Fluorescence Resonance Energy Transfer (FRET), Bacterial two Hybrid (B2H) and split Gaussia luciferase (Gluc), as well as for fusions with SNAP-tag and HaloTag for dual-color super-resolution microscopy. As proof of principle, we characterized the interaction between the Salmonella effector SipA and its chaperone InvB via split Gluc and B2H approach. The suitability for FRET analysis as well as functionality of fusions with SNAP- and HaloTag could be demonstrated by studying the transient interaction between chemotaxis response regulator CheY and its phosphatase CheZ. PMID:25856398

  17. Protein Synthesis--An Interactive Game.

    ERIC Educational Resources Information Center

    Clements, Lee Ann J.; Jackson, Karen E.

    1998-01-01

    Describes an interactive game designed to help students see and understand the dynamic relationship between DNA, RNA, and proteins. Appropriate for either a class or laboratory setting, following a lecture session about protein synthesis. (DDR)

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

    PubMed Central

    Phizicky, E M; Fields, S

    1995-01-01

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

  19. Use of protein-protein interactions in affinity chromatography.

    PubMed

    Muronetz, V I; Sholukh, M; Korpela, T

    2001-10-30

    Biospecific recognition between proteins is a phenomenon that can be exploited for designing affinity-chromatographic purification systems for proteins. In principle, the approach is straightforward, and there are usually many alternative ways, since a protein can be always found which binds specifically enough to the desired protein. Routine immunoaffinity chromatography utilizes the recognition of antigenic epitopes by antibodies. However, forces involved in protein-protein interactions as well the forces keeping the three-dimensional structures of proteins intact are complicated, and proteins are easily unfolded by various factors with unpredictable results. Because of this and because of the generally high association strength between proteins, the correct adjustment of binding forces between an immobilized protein and the protein to be purified as well as the release of bound proteins in biologically active form from affinity complexes are the main problem. Affinity systems involving interactions like enzyme-enzyme, subunit-oligomer, protein-antibody, protein-chaperone and the specific features involved in each case are presented as examples. This article also aims to sketch prospects for further development of the use of protein-protein interactions for the purification of proteins. PMID:11694271

  20. How Many Protein-Protein Interactions Types Exist in Nature?

    PubMed Central

    Mitra, Pralay; Zhang, Yang

    2012-01-01

    Protein quaternary structure universe” refers to the ensemble of all protein-protein complexes across all organisms in nature. The number of quaternary folds thus corresponds to the number of ways proteins physically interact with other proteins. This study focuses on answering two basic questions: Whether the number of protein-protein interactions is limited and, if yes, how many different quaternary folds exist in nature. By all-to-all sequence and structure comparisons, we grouped the protein complexes in the protein data bank (PDB) into 3,629 families and 1,761 folds. A statistical model was introduced to obtain the quantitative relation between the numbers of quaternary families and quaternary folds in nature. The total number of possible protein-protein interactions was estimated around 4,000, which indicates that the current protein repository contains only 42% of quaternary folds in nature and a full coverage needs approximately a quarter century of experimental effort. The results have important implications to the protein complex structural modeling and the structure genomics of protein-protein interactions. PMID:22719985

  1. How many protein-protein interactions types exist in nature?

    PubMed

    Garma, Leonardo; Mukherjee, Srayanta; Mitra, Pralay; Zhang, Yang

    2012-01-01

    "Protein quaternary structure universe" refers to the ensemble of all protein-protein complexes across all organisms in nature. The number of quaternary folds thus corresponds to the number of ways proteins physically interact with other proteins. This study focuses on answering two basic questions: Whether the number of protein-protein interactions is limited and, if yes, how many different quaternary folds exist in nature. By all-to-all sequence and structure comparisons, we grouped the protein complexes in the protein data bank (PDB) into 3,629 families and 1,761 folds. A statistical model was introduced to obtain the quantitative relation between the numbers of quaternary families and quaternary folds in nature. The total number of possible protein-protein interactions was estimated around 4,000, which indicates that the current protein repository contains only 42% of quaternary folds in nature and a full coverage needs approximately a quarter century of experimental effort. The results have important implications to the protein complex structural modeling and the structure genomics of protein-protein interactions. PMID:22719985

  2. Computational drug design targeting protein-protein interactions.

    PubMed

    Bienstock, Rachelle J

    2012-01-01

    Novel discoveries in molecular disease pathways within the cell, combined with increasing information regarding protein binding partners has lead to a new approach in drug discovery. There is interest in designing drugs to modulate protein-protein interactions as opposed to solely targeting the catalytic active site within a single enzyme or protein. There are many challenges in this new approach to drug discovery, particularly since the protein-protein interface has a larger surface area, can comprise a discontinuous epitope, and is more amorphous and less well defined than the typical drug design target, a small contained enzyme-binding pocket. Computational methods to predict modes of protein-protein interaction, as well as protein interface hot spots, have garnered significant interest, in order to facilitate the development of drugs to successfully disrupt and inhibit protein-protein interactions. This review summarizes some current methods available for computational protein-protein docking, as well as tabulating some examples of the successful design of antagonists and small molecule inhibitors for protein-protein interactions. Several of these drugs are now beginning to appear in the clinic. PMID:22316151

  3. Phytophthora infestans RXLR Effector AVR1 Interacts with Exocyst Component Sec5 to Manipulate Plant Immunity1[OPEN

    PubMed Central

    Du, Yu; Mpina, Mohamed H.; Birch, Paul R.J.; Bouwmeester, Klaas; Govers, Francine

    2015-01-01

    Phytophthora infestans secretes numerous RXLR effectors that modulate host defense and thereby pave the way for successful invasion. Here, we show that the RXLR effector AVR1 is a virulence factor that promotes colonization and suppresses callose deposition, a hallmark of basal defense. To identify host targets of AVR1, we performed yeast two-hybrid screens and selected Sec5 as a candidate. Sec5 is a subunit of the exocyst, a protein complex that is involved in vesicle trafficking. AVR1-like (A-L), a close homolog of AVR1, also acts as a virulence factor, but unlike AVR1, A-L does not suppress CRINKLER2 (CRN2)-induced cell death or interact with Sec5. Compared with AVR1, A-L is shorter and lacks the carboxyl-terminal tail, the T-region that is crucial for CRN2-induced cell death suppression and Sec5 interaction. In planta analyses revealed that AVR1 and Sec5 are in close proximity, and coimmunoprecipitation confirmed the interaction. Sec5 is required for secretion of the pathogenesis-related protein PR-1 and callose deposition and also plays a role in CRN2-induced cell death. Our findings show that P. infestans manipulates an exocyst subunit and thereby potentially disturbs vesicle trafficking, a cellular process that is important for basal defense. This is a novel strategy that oomycete pathogens exploit to modulate host defense. PMID:26336092

  4. Precise Manipulation and Patterning of Protein Crystals for Macromolecular Crystallography using Surface Acoustic Waves

    PubMed Central

    Guo, Feng; Zhou, Weijie; Li, Peng; Mao, Zhangming; Yennawar, Neela; French, Jarrod B.; Jun Huang, Tony

    2015-01-01

    Advances in modern X-ray sources and detector technology have made it possible for crystallographers to collect usable data on crystals of only a few micrometers or less in size. Despite these developments, sample handling techniques have significantly lagged behind and often prevent the full realization of current beamline capabilities. In order to address this shortcoming we have developed a surface acoustic wave-based method for manipulating and patterning crystals. This method, which does not damage the fragile protein crystals, can precisely manipulate and pattern micrometer and sub-micrometer sized crystals for data collection and screening. The technique is robust, inexpensive, and easy to implement. This method not only promises to significantly increase efficiency and throughput of both conventional and serial crystallography experiments, but also will make it possible to collect data on samples that were previously intractable. PMID:25641793

  5. Current Experimental Methods for Characterizing Protein-Protein Interactions.

    PubMed

    Zhou, Mi; Li, Qing; Wang, Renxiao

    2016-04-19

    Protein molecules often interact with other partner protein molecules in order to execute their vital functions in living organisms. Characterization of protein-protein interactions thus plays a central role in understanding the molecular mechanism of relevant protein molecules, elucidating the cellular processes and pathways relevant to health or disease for drug discovery, and charting large-scale interaction networks in systems biology research. A whole spectrum of methods, based on biophysical, biochemical, or genetic principles, have been developed to detect the time, space, and functional relevance of protein-protein interactions at various degrees of affinity and specificity. This article presents an overview of these experimental methods, outlining the principles, strengths and limitations, and recent developments of each type of method. PMID:26864455

  6. Solid State NMR and Protein-Protein Interactions in Membranes

    PubMed Central

    Miao, Yimin; Cross, Timothy A.

    2013-01-01

    Solid state NMR spectroscopy has evolved rapidly in recent years into an excellent tool for the characterization of membrane proteins and their complexes. In the past few years it has also become clear that the structure of membrane proteins, especially helical membrane proteins is determined, in part, by the membrane environment. Therefore, the modeling of this environment by a liquid crystalline lipid bilayer for solid state NMR has generated a unique tool for the characterization of native conformational states, local and global dynamics, and high resolution structure for these proteins. Protein-protein interactions can also benefit from this solid state NMR capability to characterize membrane proteins in a native-like environment. These complexes take the form of oligomeric structures and hetero-protein interactions both with water soluble proteins and other membrane proteins. PMID:24034903

  7. Solid state NMR and protein-protein interactions in membranes.

    PubMed

    Miao, Yimin; Cross, Timothy A

    2013-12-01

    Solid state NMR spectroscopy has evolved rapidly in recent years into an excellent tool for the characterization of membrane proteins and their complexes. In the past few years it has also become clear that the structure of membrane proteins, especially helical membrane proteins is determined, in part, by the membrane environment. Therefore, the modeling of this environment by a liquid crystalline lipid bilayer for solid state NMR has generated a unique tool for the characterization of native conformational states, local and global dynamics, and high-resolution structure for these proteins. Protein-protein interactions can also benefit from this solid state NMR capability to characterize membrane proteins in a native-like environment. These complexes take the form of oligomeric structures and hetero-protein interactions both with water-soluble proteins and other membrane proteins. PMID:24034903

  8. Measuring protein interactions by microchip self-interaction chromatography.

    PubMed

    García, Carlos D; Hadley, DeGail J; Wilson, W William; Henry, Charles S

    2003-01-01

    The self-interaction of proteins is of paramount importance in aggregation and crystallization phenomena. Solution conditions leading to a change in the state of aggregation of a protein, whether amorphous or crystalline, have mainly been discovered by the use of trial and error screening of large numbers of solutions. Self-interaction chromatography has the potential to provide a quantitative method for determination of protein self-interactions amenable to high-throughput screening. This paper describes the construction and characterization of a microchip separation system for low-pressure self-interaction chromatography using lysozyme as a model protein. The retention time was analyzed as a function of mobile-phase composition, amount of protein injected, flow rate, and stationary-phase modification. The capacity factors (k') as a function of crystallizing agent concentration are compared with previously published values for the osmotic second virial coefficient (B(22)) obtained by static light scattering, showing the ability of the chip to accurately determine protein-protein interactions. A 500-fold reduction in protein consumption and the possibility of using conventional instrumentation and automation are some of the advantages over currently used methodologies for evaluating protein-protein interactions. PMID:12790668

  9. A Microfluidic Platform for Characterization of Protein-Protein Interactions.

    PubMed

    Javanmard, Mehdi; Talasaz, Amirali H; Nemat-Gorgani, Mohsen; Huber, David E; Pease, Fabian; Ronaghi, Mostafa; Davis, Ronald W

    2009-08-01

    Traditionally, expensive and time consuming techniques such as mass spectrometry and Western Blotting have been used for characterization of protein-protein interactions. In this paper, we describe the design, fabrication, and testing of a rapid and inexpensive sensor, involving the use of microelectrodes in a microchannel, which can be used for real-time electrical detection of specific interactions between proteins. We have successfully demonstrated detection of target glycoprotein-glycoprotein interactions, antigen-antibody interactions, and glycoprotein-antigen interactions. We have also demonstrated the ability of this technique to distinguish between strong and weak interactions. Using this approach, it may be possible to multiplex an array of these sensors onto a chip and probe a complex mixture for various types of interactions involving protein molecules. PMID:20467571

  10. Remote Manipulator System (RMS)-based Controls-Structures Interaction (CSI) flight experiment feasibility study

    NASA Technical Reports Server (NTRS)

    Demeo, Martha E.

    1990-01-01

    The feasibility of an experiment which will provide an on-orbit validation of Controls-Structures Interaction (CSI) technology, was investigated. The experiment will demonstrate the on-orbit characterization and flexible-body control of large flexible structure dynamics using the shuttle Remote Manipulator System (RMS) with an attached payload as a test article. By utilizing existing hardware as well as establishing integration, operation and safety algorithms, techniques and procedures, the experiment will minimize the costs and risks of implementing a flight experiment. The experiment will also offer spin-off enhancement to both the Shuttle RMS (SRMS) and the Space Station RMS (SSRMS).

  11. The Intrinsic Geometric Structure of Protein-Protein Interaction Networks for Protein Interaction Prediction.

    PubMed

    Fang, Yi; Sun, Mengtian; Dai, Guoxian; Ramain, Karthik

    2016-01-01

    Recent developments in high-throughput technologies for measuring protein-protein interaction (PPI) have profoundly advanced our ability to systematically infer protein function and regulation. However, inherently high false positive and false negative rates in measurement have posed great challenges in computational approaches for the prediction of PPI. A good PPI predictor should be 1) resistant to high rate of missing and spurious PPIs, and 2) robust against incompleteness of observed PPI networks. To predict PPI in a network, we developed an intrinsic geometry structure (IGS) for network, which exploits the intrinsic and hidden relationship among proteins in network through a heat diffusion process. In this process, all explicit PPIs participate simultaneously to glue local infinitesimal and noisy experimental interaction data to generate a global macroscopic descriptions about relationships among proteins. The revealed implicit relationship can be interpreted as the probability of two proteins interacting with each other. The revealed relationship is intrinsic and robust against individual, local and explicit protein interactions in the original network. We apply our approach to publicly available PPI network data for the evaluation of the performance of PPI prediction. Experimental results indicate that, under different levels of the missing and spurious PPIs, IGS is able to robustly exploit the intrinsic and hidden relationship for PPI prediction with a higher sensitivity and specificity compared to that of recently proposed methods. PMID:26886733

  12. Geminivirus C3 Protein: Replication Enhancement and Protein Interactions

    PubMed Central

    Settlage, Sharon B.; See, Renee G.; Hanley-Bowdoin, Linda

    2005-01-01

    Most dicot-infecting geminiviruses encode a replication enhancer protein (C3, AL3, or REn) that is required for optimal replication of their small, single-stranded DNA genomes. C3 interacts with C1, the essential viral replication protein that initiates rolling circle replication. C3 also homo-oligomerizes and interacts with at least two host-encoded proteins, proliferating cell nuclear antigen (PCNA) and the retinoblastoma-related protein (pRBR). It has been proposed that protein interactions contribute to C3 function. Using the C3 protein of Tomato yellow leaf curl virus, we examined the impact of mutations to amino acids that are conserved across the C3 protein family on replication enhancement and protein interactions. Surprisingly, many of the mutations did not affect replication enhancement activity of C3 in tobacco protoplasts. Other mutations either enhanced or were detrimental to C3 replication activity. Analysis of mutated proteins in yeast two-hybrid assays indicated that mutations that inactivate C3 replication enhancement activity also reduce or inactivate C3 oligomerization and interaction with C1 and PCNA. In contrast, mutated C3 proteins impaired for pRBR binding are fully functional in replication assays. Hydrophobic residues in the middle of the C3 protein were implicated in C3 interaction with itself, C1, and PCNA, while polar resides at both the N and C termini of the protein are important for C3-pRBR interaction. These experiments established the importance of C3-C3, C3-C1, and C3-PCNA interactions in geminivirus replication. While C3-pRBR interaction is not required for viral replication in cycling cells, it may play a role during infection of differentiated cells in intact plants. PMID:16014949

  13. Protein interaction networks from literature mining

    NASA Astrophysics Data System (ADS)

    Ihara, Sigeo

    2005-03-01

    The ability to accurately predict and understand physiological changes in the biological network system in response to disease or drug therapeutics is of crucial importance in life science. The extensive amount of gene expression data generated from even a single microarray experiment often proves difficult to fully interpret and comprehend the biological significance. An increasing knowledge of protein interactions stored in the PubMed database, as well as the advancement of natural language processing, however, makes it possible to construct protein interaction networks from the gene expression information that are essential for understanding the biological meaning. From the in house literature mining system we have developed, the protein interaction network for humans was constructed. By analysis based on the graph-theoretical characterization of the total interaction network in literature, we found that the network is scale-free and semantic long-ranged interactions (i.e. inhibit, induce) between proteins dominate in the total interaction network, reducing the degree exponent. Interaction networks generated based on scientific text in which the interaction event is ambiguously described result in disconnected networks. In contrast interaction networks based on text in which the interaction events are clearly stated result in strongly connected networks. The results of protein-protein interaction networks obtained in real applications from microarray experiments are discussed: For example, comparisons of the gene expression data indicative of either a good or a poor prognosis for acute lymphoblastic leukemia with MLL rearrangements, using our system, showed newly discovered signaling cross-talk.

  14. Synthetic heparin and heparan sulfate oligosaccharides and their protein interactions.

    PubMed

    Zulueta, Medel Manuel L; Lin, Shu-Yi; Hu, Yu-Peng; Hung, Shang-Cheng

    2013-12-01

    Heparin and heparan sulfate bind a host of basic proteins that take advantage of the sugar's dense structural information. The significance of these interactions in various aspects of development, physiology, and disease stimulated keen interest in evaluating structure-activity relationships. The well-defined heparin and heparan sulfate oligosaccharides needed for these studies can be mainly accessed by chemical synthesis and, more recently by chemoenzymatic means. The various synthetic strategies available to chemical synthesis have recently enabled the acquisition of several regular and irregular sequences, including a number of dodecasaccharides, through improved coupling methods and judicial protecting group manipulations. Controlled chain elongation and critical application of modification enzymes allowed the generation of well-defined constructs via chemoenzymatic synthesis. Investigations of various protein interactions with the synthetic constructs delivered valuable information that could aid future drug development endeavors. PMID:24182748

  15. Protein-protein interactions and genetic diseases: The Interactome

    PubMed Central

    Lage, Kasper

    2014-01-01

    Protein-protein interactions mediate essentially all biological processes. Despite the quality of these data being widely questioned a decade ago, the reproducibility of large-scale protein interaction data is now much improved and there is little question that the latest screens are of high quality. Moreover, common data standards and coordinated curation practices between the databases that collect the interactions have made these valuable data available to a wide group of researchers. Here, I will review how protein-protein interactions are measured, collected and quality controlled. I discuss how the architecture of molecular protein networks have informed disease biology, and how these data are now being computationally integrated with the newest genomic technologies, in particular genome-wide association studies and exome-sequencing projects, to improve our understanding of molecular processes perturbed by genetics in human diseases. PMID:24892209

  16. Manipulating perfume delivery to the interface using polymer-surfactant interactions.

    PubMed

    Bradbury, Robert; Penfold, Jeffrey; Thomas, Robert K; Tucker, Ian M; Petkov, Jordan T; Jones, Craig

    2016-03-15

    Enhanced delivery of perfumes to interfaces is an important element of their effectiveness in a range of home and personal care products. The role of polyelectrolyte-surfactant mixtures to promote perfume adsorption at interfaces is explored here. Neutron reflectivity, NR, was used to quantify the adsorption of the model perfumes phenylethanol, PE, and linalool, LL, at the air-water interface in the presence of the anionic surfactant sodium dodecylsulfate, SDS, and the cationic polyelectrolytes, poly(dimethyldiallyl ammonium chloride), polydmdaac, and poly(ethyleneimine), PEI. The strong SDS-polydmdaac interaction dominates the surface adsorption in SDS-polymer-perfume (PE, LL) mixtures, such that the PE and LL adsorption is greatly suppressed. For PEI-SDS-perfume mixtures the PEI-LL interaction competes with the SDS-PEI interaction at all pH at the surface and significant LL adsorption occurs, whereas for PE the PEI-SDS interaction dominates and the PE adsorption is greatly reduced. The use of the strong surface polyelectrolyte-ionic surfactant interaction to manipulate perfume adsorption at the air-water interface has been demonstrated. In particular the results show how the competition between polyelectrolyte, surfactant and perfume interactions at the surface and in solution affect the partitioning of perfumes to the surface. PMID:26724705

  17. APID: Agile Protein Interaction DataAnalyzer.

    PubMed

    Prieto, Carlos; De Las Rivas, Javier

    2006-07-01

    Agile Protein Interaction DataAnalyzer (APID) is an interactive bioinformatics web tool developed to integrate and analyze in a unified and comparative platform main currently known information about protein-protein interactions demonstrated by specific small-scale or large-scale experimental methods. At present, the application includes information coming from five main source databases enclosing an unified sever to explore >35 000 different proteins and 111 000 different proven interactions. The web includes search tools to query and browse upon the data, allowing selection of the interaction pairs based in calculated parameters that weight and qualify the reliability of each given protein interaction. Such parameters are for the 'proteins': connectivity, cluster coefficient, Gene Ontology (GO) functional environment, GO environment enrichment; and for the 'interactions': number of methods, GO overlapping, iPfam domain-domain interaction. APID also includes a graphic interactive tool to visualize selected sub-networks and to navigate on them or along the whole interaction network. The application is available open access at http://bioinfow.dep.usal.es/apid/. PMID:16845013

  18. An Interactive Introduction to Protein Structure

    ERIC Educational Resources Information Center

    Lee, W. Theodore

    2004-01-01

    To improve student understanding of protein structure and the significance of noncovalent interactions in protein structure and function, students are assigned a project to write a paper complemented with computer-generated images. The assignment provides an opportunity for students to select a protein structure that is of interest and detail…

  19. Curvature-mediated interactions between membrane proteins.

    PubMed Central

    Kim, K S; Neu, J; Oster, G

    1998-01-01

    Membrane proteins can deform the lipid bilayer in which they are embedded. If the bilayer is treated as an elastic medium, then these deformations will generate elastic interactions between the proteins. The interaction between a single pair is repulsive. However, for three or more proteins, we show that there are nonpairwise forces whose magnitude is similar to the pairwise forces. When there are five or more proteins, we show that the nonpairwise forces permit the existence of stable protein aggregates, despite their pairwise repulsions. PMID:9788923

  20. Noninvasive imaging of protein-protein interactions in living animals

    NASA Astrophysics Data System (ADS)

    Luker, Gary D.; Sharma, Vijay; Pica, Christina M.; Dahlheimer, Julie L.; Li, Wei; Ochesky, Joseph; Ryan, Christine E.; Piwnica-Worms, Helen; Piwnica-Worms, David

    2002-05-01

    Protein-protein interactions control transcription, cell division, and cell proliferation as well as mediate signal transduction, oncogenic transformation, and regulation of cell death. Although a variety of methods have been used to investigate protein interactions in vitro and in cultured cells, none can analyze these interactions in intact, living animals. To enable noninvasive molecular imaging of protein-protein interactions in vivo by positron-emission tomography and fluorescence imaging, we engineered a fusion reporter gene comprising a mutant herpes simplex virus 1 thymidine kinase and green fluorescent protein for readout of a tetracycline-inducible, two-hybrid system in vivo. By using micro-positron-emission tomography, interactions between p53 tumor suppressor and the large T antigen of simian virus 40 were visualized in tumor xenografts of HeLa cells stably transfected with the imaging constructs. Imaging protein-binding partners in vivo will enable functional proteomics in whole animals and provide a tool for screening compounds targeted to specific protein-protein interactions in living animals.

  1. DIP: The Database of Interacting Proteins

    DOE Data Explorer

    The DIP Database catalogs experimentally determined interactions between proteins. It combines information from a variety of sources to create a single, consistent set of protein-protein interactions. By interaction, the DIP Database creators mean that two amino acid chains were experimentally identified to bind to each other. The database lists such pairs to aid those studying a particular protein-protein interaction but also those investigating entire regulatory and signaling pathways as well as those studying the organisation and complexity of the protein interaction network at the cellular level. The data stored within the DIP database were curated, both, manually by expert curators and also automatically using computational approaches that utilize the knowledge about the protein-protein interaction networks extracted from the most reliable, core subset of the DIP data. It is a relational database that can be searched by protein, sequence, motif, article information, and pathBLAST. The website also serves as an access point to a number of projects related to DIP, such as LiveDIP, The Database of Ligand-Receptor Partners (DLRP) and JDIP. Users have free and open access to DIP after login. [Taken from the DIP Guide and the DIP website] (Specialized Interface) (Registration Required)

  2. 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

  3. Understanding and Manipulating Electrostatic Fields at the Protein-Protein Interface Using Vibrational Spectroscopy and Continuum Electrostatics Calculations.

    PubMed

    Ritchie, Andrew W; Webb, Lauren J

    2015-11-01

    Biological function emerges in large part from the interactions of biomacromolecules in the complex and dynamic environment of the living cell. For this reason, macromolecular interactions in biological systems are now a major focus of interest throughout the biochemical and biophysical communities. The affinity and specificity of macromolecular interactions are the result of both structural and electrostatic factors. Significant advances have been made in characterizing structural features of stable protein-protein interfaces through the techniques of modern structural biology, but much less is understood about how electrostatic factors promote and stabilize specific functional macromolecular interactions over all possible choices presented to a given molecule in a crowded environment. In this Feature Article, we describe how vibrational Stark effect (VSE) spectroscopy is being applied to measure electrostatic fields at protein-protein interfaces, focusing on measurements of guanosine triphosphate (GTP)-binding proteins of the Ras superfamily binding with structurally related but functionally distinct downstream effector proteins. In VSE spectroscopy, spectral shifts of a probe oscillator's energy are related directly to that probe's local electrostatic environment. By performing this experiment repeatedly throughout a protein-protein interface, an experimental map of measured electrostatic fields generated at that interface is determined. These data can be used to rationalize selective binding of similarly structured proteins in both in vitro and in vivo environments. Furthermore, these data can be used to compare to computational predictions of electrostatic fields to explore the level of simulation detail that is necessary to accurately predict our experimental findings. PMID:26375183

  4. Evolutionarily Conserved Herpesviral Protein Interaction Networks

    PubMed Central

    Fossum, Even; Friedel, Caroline C.; Rajagopala, Seesandra V.; Titz, Björn; Baiker, Armin; Schmidt, Tina; Kraus, Theo; Stellberger, Thorsten; Rutenberg, Christiane; Suthram, Silpa; Bandyopadhyay, Sourav; Rose, Dietlind; von Brunn, Albrecht; Uhlmann, Mareike; Zeretzke, Christine; Dong, Yu-An; Boulet, Hélène; Koegl, Manfred; Bailer, Susanne M.; Koszinowski, Ulrich; Ideker, Trey; Uetz, Peter; Zimmer, Ralf; Haas, Jürgen

    2009-01-01

    Herpesviruses constitute a family of large DNA viruses widely spread in vertebrates and causing a variety of different diseases. They possess dsDNA genomes ranging from 120 to 240 kbp encoding between 70 to 170 open reading frames. We previously reported the protein interaction networks of two herpesviruses, varicella-zoster virus (VZV) and Kaposi's sarcoma-associated herpesvirus (KSHV). In this study, we systematically tested three additional herpesvirus species, herpes simplex virus 1 (HSV-1), murine cytomegalovirus and Epstein-Barr virus, for protein interactions in order to be able to perform a comparative analysis of all three herpesvirus subfamilies. We identified 735 interactions by genome-wide yeast-two-hybrid screens (Y2H), and, together with the interactomes of VZV and KSHV, included a total of 1,007 intraviral protein interactions in the analysis. Whereas a large number of interactions have not been reported previously, we were able to identify a core set of highly conserved protein interactions, like the interaction between HSV-1 UL33 with the nuclear egress proteins UL31/UL34. Interactions were conserved between orthologous proteins despite generally low sequence similarity, suggesting that function may be more conserved than sequence. By combining interactomes of different species we were able to systematically address the low coverage of the Y2H system and to extract biologically relevant interactions which were not evident from single species. PMID:19730696

  5. PLIC: protein-ligand interaction clusters.

    PubMed

    Anand, Praveen; Nagarajan, Deepesh; Mukherjee, Sumanta; Chandra, Nagasuma

    2014-01-01

    Most of the biological processes are governed through specific protein-ligand interactions. Discerning different components that contribute toward a favorable protein- ligand interaction could contribute significantly toward better understanding protein function, rationalizing drug design and obtaining design principles for protein engineering. The Protein Data Bank (PDB) currently hosts the structure of ∼68 000 protein-ligand complexes. Although several databases exist that classify proteins according to sequence and structure, a mere handful of them annotate and classify protein-ligand interactions and provide information on different attributes of molecular recognition. In this study, an exhaustive comparison of all the biologically relevant ligand-binding sites (84 846 sites) has been conducted using PocketMatch: a rapid, parallel, in-house algorithm. PocketMatch quantifies the similarity between binding sites based on structural descriptors and residue attributes. A similarity network was constructed using binding sites whose PocketMatch scores exceeded a high similarity threshold (0.80). The binding site similarity network was clustered into discrete sets of similar sites using the Markov clustering (MCL) algorithm. Furthermore, various computational tools have been used to study different attributes of interactions within the individual clusters. The attributes can be roughly divided into (i) binding site characteristics including pocket shape, nature of residues and interaction profiles with different kinds of atomic probes, (ii) atomic contacts consisting of various types of polar, hydrophobic and aromatic contacts along with binding site water molecules that could play crucial roles in protein-ligand interactions and (iii) binding energetics involved in interactions derived from scoring functions developed for docking. For each ligand-binding site in each protein in the PDB, site similarity information, clusters they belong to and description of

  6. Real-time interactive visualization and manipulation of the volumetric data using GPU-based methods

    NASA Astrophysics Data System (ADS)

    Dietrich, Carlos A.; Nedel, Luciana P.; Olabarriaga, Silvia D.; Comba, Joao L. D.; Zanchet, Dinamar J.; Marques da Silva, Ana M.; de Souza Montero, Edna F.

    2004-05-01

    This work presents a set of tools developed to provide 3D visualization and interaction with large volumetric data that relies on recent programmable capabilities of consumer-level graphics cards. We are exploiting the programmable control of calculations performed by the graphics hardware for generating the appearance of each pixel on the screen to develop real-time, interactive volume manipulation tools. These tools allow real-time modification of visualization parameters, such as color and opacity classification or the selection of a volume of interest, extending the benefit of hardware acceleration beyond display, namely for computation of voxel visibility. Three interactive tools are proposed: a cutting tool that allows the selection of a convex volume of interest, an eraser-like tool to eliminate non-relevant parts of the image and a digger-like tool that allows the user to eliminate layers of a 3D image. To interactively apply the proposed tools on a volume, we are making use of some so known user interaction techniques, as the ones used in 2D painting systems. Our strategy is to minimize the user entrainment efforts involved in the tools learning. Finally, we illustrate the potential application of the conceived tools for preoperative planning of liver surgery and for liver vascular anatomy study. Preliminary results concerning the system performance and the images quality and resolution are presented and discussed.

  7. PPIM: A Protein-Protein Interaction Database for Maize.

    PubMed

    Zhu, Guanghui; Wu, Aibo; Xu, Xin-Jian; Xiao, Pei-Pei; Lu, Le; Liu, Jingdong; Cao, Yongwei; Chen, Luonan; Wu, Jun; Zhao, Xing-Ming

    2016-02-01

    Maize (Zea mays) is one of the most important crops worldwide. To understand the biological processes underlying various traits of the crop (e.g. yield and response to stress), a detailed protein-protein interaction (PPI) network is highly demanded. Unfortunately, there are very few such PPIs available in the literature. Therefore, in this work, we present the Protein-Protein Interaction Database for Maize (PPIM), which covers 2,762,560 interactions among 14,000 proteins. The PPIM contains not only accurately predicted PPIs but also those molecular interactions collected from the literature. The database is freely available at http://comp-sysbio.org/ppim with a user-friendly powerful interface. We believe that the PPIM resource can help biologists better understand the maize crop. PMID:26620522

  8. 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

  9. Manipulating the Prion Protein Gene Sequence and Expression Levels with CRISPR/Cas9

    PubMed Central

    Kaczmarczyk, Lech; Mende, Ylva; Zevnik, Branko; Jackson, Walker S.

    2016-01-01

    The mammalian prion protein (PrP, encoded by Prnp) is most infamous for its central role in prion diseases, invariably fatal neurodegenerative diseases affecting humans, food animals, and animals in the wild. However, PrP is also hypothesized to be an important receptor for toxic protein conformers in Alzheimer's disease, and is associated with other clinically relevant processes such as cancer and stroke. Thus, key insights into important clinical areas, as well as into understanding PrP functions in normal physiology, can be obtained from studying transgenic mouse models and cell culture systems. However, the Prnp locus is difficult to manipulate by homologous recombination, making modifications of the endogenous locus rarely attempted. Fortunately in recent years genome engineering technologies, like TALENs or CRISPR/Cas9 (CC9), have brought exceptional new possibilities for manipulating Prnp. Herein, we present our observations made during systematic experiments with the CC9 system targeting the endogenous mouse Prnp locus, to either modify sequences or to boost PrP expression using CC9-based synergistic activation mediators (SAMs). It is our hope that this information will aid and encourage researchers to implement gene-targeting techniques into their research program. PMID:27128441

  10. Manipulating the Prion Protein Gene Sequence and Expression Levels with CRISPR/Cas9.

    PubMed

    Kaczmarczyk, Lech; Mende, Ylva; Zevnik, Branko; Jackson, Walker S

    2016-01-01

    The mammalian prion protein (PrP, encoded by Prnp) is most infamous for its central role in prion diseases, invariably fatal neurodegenerative diseases affecting humans, food animals, and animals in the wild. However, PrP is also hypothesized to be an important receptor for toxic protein conformers in Alzheimer's disease, and is associated with other clinically relevant processes such as cancer and stroke. Thus, key insights into important clinical areas, as well as into understanding PrP functions in normal physiology, can be obtained from studying transgenic mouse models and cell culture systems. However, the Prnp locus is difficult to manipulate by homologous recombination, making modifications of the endogenous locus rarely attempted. Fortunately in recent years genome engineering technologies, like TALENs or CRISPR/Cas9 (CC9), have brought exceptional new possibilities for manipulating Prnp. Herein, we present our observations made during systematic experiments with the CC9 system targeting the endogenous mouse Prnp locus, to either modify sequences or to boost PrP expression using CC9-based synergistic activation mediators (SAMs). It is our hope that this information will aid and encourage researchers to implement gene-targeting techniques into their research program. PMID:27128441

  11. Capturing the Interaction Potential of Amyloidogenic Proteins

    SciTech Connect

    Javid, Nadeem; Vogtt, Karsten; Winter, Roland; Krywka, Christina; Tolan, Metin

    2007-07-13

    Experimentally derived static structure factors obtained for the aggregation-prone protein insulin were analyzed with a statistical mechanical model based on the Derjaguin-Landau-Verwey-Overbeek potential. The data reveal that the protein self-assembles into equilibrium clusters already at low concentrations. Furthermore, striking differences regarding interaction forces between aggregation-prone proteins such as insulin in the preaggregated regime and natively stable globular proteins are found.

  12. Methods for analyzing and quantifying protein-protein interaction.

    PubMed

    Syafrizayanti; Betzen, Christian; Hoheisel, Jörg D; Kastelic, Damjana

    2014-02-01

    Genome sequencing has led to the identification of many proteins, which had not been recognized before. In consequence, the basic set of human proteins is generally known. Far less information, however, exists about protein-protein interactions, which are required and responsible for cellular activities and their control. Many protein isoforms that result from mutations, splice-variations and post-translational modifications also come into play. Until recently, interactions of only few protein partners could be analyzed in a single experiment. However, this does not meet the challenge of investigating the highly complex interaction patterns in cellular systems. It is made even more demanding by the need to determine the intensity of interactions quantitatively in order to properly understand protein interplay. Currently available techniques vary with respect to accuracy, reliability, reproducibility and throughput and their performances range from a mere qualitative demonstration of binding to a quantitative characterization of affinities. In this article, an overview is given of the methodologies available for analysis of protein-protein interactions. PMID:24393018

  13. Signature Product Code for Predicting Protein-Protein Interactions

    SciTech Connect

    Martin, Shawn B.; Brown, William M.

    2004-09-25

    The SigProdV1.0 software consists of four programs which together allow the prediction of protein-protein interactions using only amino acid sequences and experimental data. The software is based on the use of tensor products of amino acid trimers coupled with classifiers known as support vector machines. Essentially the program looks for amino acid trimer pairs which occur more frequently in protein pairs which are known to interact. These trimer pairs are then used to make predictions about unknown protein pairs. A detailed description of the method can be found in the paper: S. Martin, D. Roe, J.L. Faulon. "Predicting protein-protein interactions using signature products," Bioinformatics, available online from Advance Access, Aug. 19, 2004.

  14. Signature Product Code for Predicting Protein-Protein Interactions

    Energy Science and Technology Software Center (ESTSC)

    2004-09-25

    The SigProdV1.0 software consists of four programs which together allow the prediction of protein-protein interactions using only amino acid sequences and experimental data. The software is based on the use of tensor products of amino acid trimers coupled with classifiers known as support vector machines. Essentially the program looks for amino acid trimer pairs which occur more frequently in protein pairs which are known to interact. These trimer pairs are then used to make predictionsmore » about unknown protein pairs. A detailed description of the method can be found in the paper: S. Martin, D. Roe, J.L. Faulon. "Predicting protein-protein interactions using signature products," Bioinformatics, available online from Advance Access, Aug. 19, 2004.« less

  15. Quantitative interaction proteomics of neurodegenerative disease proteins.

    PubMed

    Hosp, Fabian; Vossfeldt, Hannes; Heinig, Matthias; Vasiljevic, Djordje; Arumughan, Anup; Wyler, Emanuel; Landthaler, Markus; Hubner, Norbert; Wanker, Erich E; Lannfelt, Lars; Ingelsson, Martin; Lalowski, Maciej; Voigt, Aaron; Selbach, Matthias

    2015-05-19

    Several proteins have been linked to neurodegenerative disorders (NDDs), but their molecular function is not completely understood. Here, we used quantitative interaction proteomics to identify binding partners of Amyloid beta precursor protein (APP) and Presenilin-1 (PSEN1) for Alzheimer's disease (AD), Huntingtin (HTT) for Huntington's disease, Parkin (PARK2) for Parkinson's disease, and Ataxin-1 (ATXN1) for spinocerebellar ataxia type 1. Our network reveals common signatures of protein degradation and misfolding and recapitulates known biology. Toxicity modifier screens and comparison to genome-wide association studies show that interaction partners are significantly linked to disease phenotypes in vivo. Direct comparison of wild-type proteins and disease-associated variants identified binders involved in pathogenesis, highlighting the value of differential interactome mapping. Finally, we show that the mitochondrial protein LRPPRC interacts preferentially with an early-onset AD variant of APP. This interaction appears to induce mitochondrial dysfunction, which is an early phenotype of AD. PMID:25959826

  16. Protein interaction mapping: A Drosophila case study

    PubMed Central

    Formstecher, Etienne; Aresta, Sandra; Collura, Vincent; Hamburger, Alexandre; Meil, Alain; Trehin, Alexandra; Reverdy, Céline; Betin, Virginie; Maire, Sophie; Brun, Christine; Jacq, Bernard; Arpin, Monique; Bellaiche, Yohanns; Bellusci, Saverio; Benaroch, Philippe; Bornens, Michel; Chanet, Roland; Chavrier, Philippe; Delattre, Olivier; Doye, Valérie; Fehon, Richard; Faye, Gérard; Galli, Thierry; Girault, Jean-Antoine; Goud, Bruno; de Gunzburg, Jean; Johannes, Ludger; Junier, Marie-Pierre; Mirouse, Vincent; Mukherjee, Ashim; Papadopoulo, Dora; Perez, Franck; Plessis, Anne; Rossé, Carine; Saule, Simon; Stoppa-Lyonnet, Dominique; Vincent, Alain; White, Michael; Legrain, Pierre; Wojcik, Jérôme; Camonis, Jacques; Daviet, Laurent

    2005-01-01

    The Drosophila (fruit fly) model system has been instrumental in our current understanding of human biology, development, and diseases. Here, we used a high-throughput yeast two-hybrid (Y2H)-based technology to screen 102 bait proteins from Drosophila melanogaster, most of them orthologous to human cancer-related and/or signaling proteins, against high-complexity fly cDNA libraries. More than 2300 protein-protein interactions (PPI) were identified, of which 710 are of high confidence. The computation of a reliability score for each protein-protein interaction and the systematic identification of the interacting domain combined with a prediction of structural/functional motifs allow the elaboration of known complexes and the identification of new ones. The full data set can be visualized using a graphical Web interface, the PIMRider (http://pim.hybrigenics.com), and is also accessible in the PSI standard Molecular Interaction data format. Our fly Protein Interaction Map (PIM) is surprisingly different from the one recently proposed by Giot et al. with little overlap between the two data sets. Analysis of the differences in data sets and methods suggests alternative strategies to enhance the accuracy and comprehensiveness of the post-genomic generation of broad-scale protein interaction maps. PMID:15710747

  17. Retention and interference of learned dexterous manipulation: interaction between multiple sensorimotor processes.

    PubMed

    Fu, Qiushi; Santello, Marco

    2015-01-01

    An object can be used in multiple contexts, each requiring different hand actions. How the central nervous system builds and maintains memory of such dexterous manipulations remains unclear. We conducted experiments in which human subjects had to learn and recall manipulations performed in two contexts, A and B. Both contexts involved lifting the same L-shaped object whose geometry cued its asymmetrical mass distribution. Correct performance required producing a torque on the vertical handle at object lift onset to prevent it from tilting. The torque direction depended on the context, i.e., object orientation, which was changed by 180° object rotation about a vertical axis. With an A1B1A2 context switching paradigm, subjects learned A1 in the first block of eight trials as indicated by a torque approaching the required one. However, subjects made large errors in anticipating the required torque when switching to B1 immediately after A1 (negative transfer), as well as when they had to recall A1 when switching to A2 after learning B through another block of eight lifts (retrieval interference). Classic sensorimotor learning theories attribute such interferences to multi-rate, multi-state error-driven updates of internal models. However, by systematically changing the interblock break duration and within-block number of trials, our results suggest an alternative explanation underlying interference and retention of dexterous manipulation. Specifically, we identified and quantified through a novel computational model the nonlinear interaction between two sensorimotor mechanisms: a short-lived, context-independent, use-dependent sensorimotor memory and a context-sensitive, error-based learning process. PMID:25274349

  18. Sitewise manipulations and Mott insulator-superfluid transition of interacting photons using superconducting circuit simulators

    DOE PAGESBeta

    Deng, Xiuhao; Jia, Chunjing; Chien, Chih-Chun

    2015-02-23

    We report that the Bose Hubbard model (BHM) of interacting bosons in a lattice has been a paradigm in many-body physics, and it exhibits a Mott insulator (MI)-superfluid (SF) transition at integer filling. Here a quantum simulator of the BHM using a superconducting circuit is proposed. Specifically, a superconducting transmission line resonator supporting microwave photons is coupled to a charge qubit to form one site of the BHM, and adjacent sites are connected by a tunable coupler. To obtain a mapping from the superconducting circuit to the BHM, we focus on the dispersive regime where the excitations remain photonlike. Standardmore » perturbation theory is implemented to locate the parameter range where the MI-SF transition may be simulated. This simulator allows single-site manipulations and we illustrate this feature by considering two scenarios where a single-site manipulation can drive a MI-SF transition. The transition can be analyzed by mean-field analyses, and the exact diagonalization was implemented to provide accurate results. The variance of the photon density and the fidelity metric clearly show signatures of the transition. Lastly, experimental realizations and other possible applications of this simulator are also discussed.« less

  19. Sitewise manipulations and Mott insulator-superfluid transition of interacting photons using superconducting circuit simulators

    SciTech Connect

    Deng, Xiuhao; Jia, Chunjing; Chien, Chih-Chun

    2015-02-23

    We report that the Bose Hubbard model (BHM) of interacting bosons in a lattice has been a paradigm in many-body physics, and it exhibits a Mott insulator (MI)-superfluid (SF) transition at integer filling. Here a quantum simulator of the BHM using a superconducting circuit is proposed. Specifically, a superconducting transmission line resonator supporting microwave photons is coupled to a charge qubit to form one site of the BHM, and adjacent sites are connected by a tunable coupler. To obtain a mapping from the superconducting circuit to the BHM, we focus on the dispersive regime where the excitations remain photonlike. Standard perturbation theory is implemented to locate the parameter range where the MI-SF transition may be simulated. This simulator allows single-site manipulations and we illustrate this feature by considering two scenarios where a single-site manipulation can drive a MI-SF transition. The transition can be analyzed by mean-field analyses, and the exact diagonalization was implemented to provide accurate results. The variance of the photon density and the fidelity metric clearly show signatures of the transition. Lastly, experimental realizations and other possible applications of this simulator are also discussed.

  20. Multilevel regulation of protein protein interactions in biological circuitry

    NASA Astrophysics Data System (ADS)

    Beckett, Dorothy

    2005-06-01

    Protein-protein interactions are central to biology and, in this 'post-genomic era', prediction of these interactions has become the goal of many computational efforts. Close inspection of even relatively simple biological regulatory circuitry reveals multiple levels of control of the contributing protein interactions. The fundamental probability that an interaction will occur under a given set of conditions is difficult to predict because the relationship between structure and energy is not known. Layered on this basic difficulty are allosteric control mechanisms involving post-translational modification or small ligand binding. In addition, many biological processes involve multiple protein-protein interactions, some of which may be cooperative or even competitive. Finally, although the emphasis in predicting protein interactions is based on equilibrium thermodynamic principles, kinetics can be a major controlling feature in these systems. This complexity reinforces the necessity of performing detailed quantitative studies of the component interactions of complex biological regulatory systems. Results of such studies will help us to bridge the gap between our knowledge of structure and our understanding of functional biology.

  1. Characterizing carbohydrate-protein interactions by NMR

    PubMed Central

    Bewley, Carole A.; Shahzad-ul-Hussan, Syed

    2013-01-01

    Interactions between proteins and soluble carbohydrates and/or surface displayed glycans are central to countless recognition, attachment and signaling events in biology. The physical chemical features associated with these binding events vary considerably, depending on the biological system of interest. For example, carbohydrate-protein interactions can be stoichiometric or multivalent, the protein receptors can be monomeric or oligomeric, and the specificity of recognition can be highly stringent or rather promiscuous. Equilibrium dissociation constants for carbohydrate binding are known to vary from micromolar to millimolar, with weak interactions being far more prevalent; and individual carbohydrate binding sites can be truly symmetrical or merely homologous, and hence, the affinities of individual sites within a single protein can vary, as can the order of binding. Several factors, including the weak affinities with which glycans bind their protein receptors, the dynamic nature of the glycans themselves, and the non-equivalent interactions among oligomeric carbohydrate receptors, have made NMR an especially powerful tool for studying and defining carbohydrate-protein interactions. Here we describe those NMR approaches that have proven to be the most robust in characterizing these systems, and explain what type of information can (or cannot) be obtained from each. Our goal is to provide to the reader the information necessary for selecting the correct experiment or sets of experiments to characterize their carbohydrate-protein interaction of interest. PMID:23784792

  2. Van der Waals Interactions Involving Proteins

    NASA Technical Reports Server (NTRS)

    Roth, Charles M.; Neal, Brian L.; Lenhoff, Abraham M.

    1996-01-01

    Van der Waals (dispersion) forces contribute to interactions of proteins with other molecules or with surfaces, but because of the structural complexity of protein molecules, the magnitude of these effects is usually estimated based on idealized models of the molecular geometry, e.g., spheres or spheroids. The calculations reported here seek to account for both the geometric irregularity of protein molecules and the material properties of the interacting media. Whereas the latter are found to fall in the generally accepted range, the molecular shape is shown to cause the magnitudes of the interactions to differ significantly from those calculated using idealized models. with important consequences. First, the roughness of the molecular surface leads to much lower average interaction energies for both protein-protein and protein-surface cases relative to calculations in which the protein molecule is approximated as a sphere. These results indicate that a form of steric stabilization may be an important effect in protein solutions. Underlying this behavior is appreciable orientational dependence, one reflection of which is that molecules of complementary shape are found to exhibit very strong attractive dispersion interactions. Although this has been widely discussed previously in the context of molecular recognition processes, the broader implications of these phenomena may also be important at larger molecular separations, e.g., in the dynamics of aggregation, precipitation, and crystal growth.

  3. Van der Waals interactions involving proteins.

    PubMed Central

    Roth, C M; Neal, B L; Lenhoff, A M

    1996-01-01

    Van der Waals (dispersion) forces contribute to interactions of proteins with other molecules or with surfaces, but because of the structural complexity of protein molecules, the magnitude of these effects is usually estimated based on idealized models of the molecular geometry, e.g., spheres or spheroids. The calculations reported here seek to account for both the geometric irregularity of protein molecules and the material properties of the interacting media. Whereas the latter are found to fall in the generally accepted range, the molecular shape is shown to cause the magnitudes of the interactions to differ significantly from those calculated using idealized models, with important consequences. First, the roughness of the molecular surface leads to much lower average interaction energies for both protein-protein and protein-surface cases relative to calculations in which the protein molecule is approximated as a sphere. These results indicate that a form of steric stabilization may be an important effect in protein solutions. Underlying this behavior is appreciable orientational dependence, one reflection of which is that molecules of complementary shape are found to exhibit very strong attractive dispersion interactions. Although this has been widely discussed previously in the context of molecular recognition processes, the broader implications of these phenomena may also be important at larger molecular separations, e.g., in the dynamics of aggregation, precipitation, and crystal growth. Images FIGURE 3 PMID:8789115

  4. FAK and p53 protein interactions.

    PubMed

    Golubovskaya, Vita M; Cance, William G

    2011-09-01

    Focal Adhesion Kinase plays a major role in cell adhesion, motility, survival, proliferation, metastasis, angiogenesis and lymphangiogenesis. In 2004, we have cloned the promoter sequence of FAK and found that p53 inhibits its activity (BBA, v. 1678, 2004). In 2005, we were the first group to show that FAK and p53 proteins directly interact in the cells (JBC, v. 280, 2005). We have shown that FAK and p53 proteins interact in the cytoplasm and in the nucleus by immunoprecipitation, pull-down and confocal microscopy assays. We have shown that FAK inhibited activity of p53 with the transcriptional targets: p21, Bax and Mdm-2 through protein-protein interactions. We identified the 7 amino-acid site in p53 that is involved in interaction with FAK protein. The present review will discuss the interaction of FAK and p53 proteins and discuss the mechanism of FAK-p53 loop regulation: inhibition of FAK promoter activity by p53 protein and also inhibition of p53 transcriptional activity by FAK protein. PMID:21355845

  5. RNA Protein Interaction in Neurons

    PubMed Central

    Darnell, Robert B.

    2013-01-01

    Neurons have their own systems for regulating RNA. Several multigene families encode RNA binding proteins (RNABPs) that are uniquely expressed in neurons, including the well-known neuron-specific markers ELAV and NeuN, and the disease antigen NOVA. New technologies have emerged in recent years to assess the function of these proteins in vivo, and the answers are yielding insights into how and why neurons may regulate RNA in special ways—to increase cellular complexity, to spatially localize mRNA, and to regulate their expression in response to synaptic stimuli. The functions of such restricted neuronal proteins is likely to be complimented by more widely expressed RNABPs that may themselves have developed specialized functions in neurons, including Argonaute/miRNAs. Here we review what is known about such RNABPs, and explore the potential biologic and neurologic significance of neuronal RNA regulatory systems. PMID:23701460

  6. Contribution of Hydrophobic Interactions to Protein Stability

    PubMed Central

    Pace, C. Nick; Fu, Hailong; Fryar, Katrina Lee; Landua, John; Trevino, Saul R.; Shirley, Bret A.; Hendricks, Marsha McNutt; Iimura, Satoshi; Gajiwala, Ketan; Scholtz, J. Martin; Grimsley, Gerald R.

    2011-01-01

    Our goal was to gain a better understanding of the contribution of hydrophobic interactions to protein stability. We measured the change in conformational stability, Δ(ΔG), for hydrophobic mutants of four proteins: villin head piece subdomain (VHP) with 36 residues, a surface protein from Borrelia burgdorferi (VlsE) with 341 residues, and two proteins previously studied in our laboratory, ribonucleases Sa and T1. We compare our results with previous studies and reach the following conclusions. 1. Hydrophobic interactions contribute less to the stability of a small protein, VHP (0.6 ± 0.3 kcal/mole per –CH2– group), than to the stability of a large protein, VlsE (1.6 ± 0.3 kcal/mol per –CH2– group). 2. Hydrophobic interactions make the major contribution to the stability of VHP (40 kcal/mol) and the major contributors are (in kcal/mol): Phe 18 (3.9), Met 13 (3.1), Phe 7 (2.9), Phe 11 (2.7), and Leu 21 (2.7). 3. Based on Δ(ΔG) values for 148 hydrophobic mutants in 13 proteins, burying a –CH2– group on folding contributes, on average, 1.1 ± 0.5 kcal/mol to protein stability. 4. The experimental Δ(ΔG) values for aliphatic side chains (Ala, Val, Ile, and Leu) are in good agreement with their ΔGtr values from water to cyclohexane. 5. For 22 proteins with 36 to 534 residues, hydrophobic interactions contribute 60 ± 4% and hydrogen bonds 40 ± 4% to protein stability. 6. Conformational entropy contributes about 2.4 kcal/mol per residue to protein instability. The globular conformation of proteins is stabilized predominately by hydrophobic interactions. PMID:21377472

  7. Contribution of hydrophobic interactions to protein stability.

    PubMed

    Pace, C Nick; Fu, Hailong; Fryar, Katrina Lee; Landua, John; Trevino, Saul R; Shirley, Bret A; Hendricks, Marsha McNutt; Iimura, Satoshi; Gajiwala, Ketan; Scholtz, J Martin; Grimsley, Gerald R

    2011-05-01

    Our goal was to gain a better understanding of the contribution of hydrophobic interactions to protein stability. We measured the change in conformational stability, Δ(ΔG), for hydrophobic mutants of four proteins: villin headpiece subdomain (VHP) with 36 residues, a surface protein from Borrelia burgdorferi (VlsE) with 341 residues, and two proteins previously studied in our laboratory, ribonucleases Sa and T1. We compared our results with those of previous studies and reached the following conclusions: (1) Hydrophobic interactions contribute less to the stability of a small protein, VHP (0.6±0.3 kcal/mol per -CH(2)- group), than to the stability of a large protein, VlsE (1.6±0.3 kcal/mol per -CH(2)- group). (2) Hydrophobic interactions make the major contribution to the stability of VHP (40 kcal/mol) and the major contributors are (in kilocalories per mole) Phe18 (3.9), Met13 (3.1), Phe7 (2.9), Phe11 (2.7), and Leu21 (2.7). (3) Based on the Δ(ΔG) values for 148 hydrophobic mutants in 13 proteins, burying a -CH(2)- group on folding contributes, on average, 1.1±0.5 kcal/mol to protein stability. (4) The experimental Δ(ΔG) values for aliphatic side chains (Ala, Val, Ile, and Leu) are in good agreement with their ΔG(tr) values from water to cyclohexane. (5) For 22 proteins with 36 to 534 residues, hydrophobic interactions contribute 60±4% and hydrogen bonds contribute 40±4% to protein stability. (6) Conformational entropy contributes about 2.4 kcal/mol per residue to protein instability. The globular conformation of proteins is stabilized predominantly by hydrophobic interactions. PMID:21377472

  8. Interface-Resolved Network of Protein-Protein Interactions

    PubMed Central

    Johnson, Margaret E.; Hummer, Gerhard

    2013-01-01

    We define an interface-interaction network (IIN) to capture the specificity and competition between protein-protein interactions (PPI). This new type of network represents interactions between individual interfaces used in functional protein binding and thereby contains the detail necessary to describe the competition and cooperation between any pair of binding partners. Here we establish a general framework for the construction of IINs that merges computational structure-based interface assignment with careful curation of available literature. To complement limited structural data, the inclusion of biochemical data is critical for achieving the accuracy and completeness necessary to analyze the specificity and competition between the protein interactions. Firstly, this procedure provides a means to clarify the information content of existing data on purported protein interactions and to remove indirect and spurious interactions. Secondly, the IIN we have constructed here for proteins involved in clathrin-mediated endocytosis (CME) exhibits distinctive topological properties. In contrast to PPI networks with their global and relatively dense connectivity, the fragmentation of the IIN into distinctive network modules suggests that different functional pressures act on the evolution of its topology. Large modules in the IIN are formed by interfaces sharing specificity for certain domain types, such as SH3 domains distributed across different proteins. The shared and distinct specificity of an interface is necessary for effective negative and positive design of highly selective binding targets. Lastly, the organization of detailed structural data in a network format allows one to identify pathways of specific binding interactions and thereby predict effects of mutations at specific surfaces on a protein and of specific binding inhibitors, as we explore in several examples. Overall, the endocytosis IIN is remarkably complex and rich in features masked in the coarser

  9. Moonlighting proteins in sperm-egg interactions.

    PubMed

    Petit, François M; Serres, Catherine; Auer, Jana

    2014-12-01

    Sperm-egg interaction is a highly species-specific step during the fertilization process. The first steps consist of recognition between proteins on the sperm head and zona pellucida (ZP) glycoproteins, the acellular coat that protects the oocyte. We aimed to determine which sperm head proteins interact with ZP2, ZP3 and ZP4 in humans. Two approaches were combined to identify these proteins: immunoblotting human spermatozoa targeted by antisperm antibodies (ASAs) from infertile men and far-Western blotting of human sperm proteins overlaid by each of the human recombinant ZP (hrZP) proteins. We used a proteomic approach with 2D electrophoretic separation of sperm protein revealed using either ASAs eluted from infertile patients or recombinant human ZP glycoproteins expressed in Chinese-hamster ovary (CHO) cells. Only spots highlighted by both methods were analysed by MALDI-MS/MS for identification. We identified proteins already described in human spermatozoa, but implicated in different metabolic pathways such as glycolytic enzymes [phosphokinase type 3 (PK3), enolase 1 (ENO1), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), aldolase A (ALDOA) and triose phosphate isomerase (TPI)], detoxification enzymes [GST Mu (GSTM) and phospholipid hydroperoxide glutathione peroxidase (PHGPx) 4], ion channels [voltage-dependent anion channel 2 (VDAC2)] or structural proteins (outer dense fibre 2). Several proteins were localized on the sperm head by indirect immunofluorescence, and their interaction with ZP proteins was confirmed by co-precipitation experiments. These results confirm the complexity of the sperm-ZP recognition process in humans with the implication of different proteins interacting with the main three ZP glycoproteins. The multiple roles of these proteins suggest that they are multifaceted or moonlighting proteins. PMID:25399599

  10. Effects of surface compositional and structural heterogeneity on nanoparticle-protein interactions: different protein configurations.

    PubMed

    Huang, Rixiang; Carney, Randy P; Ikuma, Kaoru; Stellacci, Francesco; Lau, Boris L T

    2014-06-24

    As nanoparticles (NPs) enter into biological systems, they are immediately exposed to a variety and concentration of proteins. The physicochemical interactions between proteins and NPs are influenced by the surface properties of the NPs. To identify the effects of NP surface heterogeneity, the interactions between bovine serum albumin (BSA) and gold NPs (AuNPs) with similar chemical composition but different surface structures were investigated. Different interaction modes and BSA conformations were studied by dynamic light scattering, circular dichroism spectroscopy, fluorescence quenching and isothermal titration calorimetry (ITC). Depending on the surface structure of AuNPs, BSA seems to adopt either a "side-on" or an "end-on" conformation on AuNPs. ITC demonstrated that the adsorption of BSA onto AuNPs with randomly distributed polar and nonpolar groups was primarily driven by electrostatic interaction, and all BSA were adsorbed in the same process. The adsorption of BSA onto AuNPs covered with alternating domains of polar and nonpolar groups was a combination of different interactions. Overall, the results of this study point to the potential for utilizing nanoscale manipulation of NP surfaces to control the resulting NP-protein interactions. PMID:24882660

  11. Website on Protein Interaction and Protein Structure Related Work

    NASA Technical Reports Server (NTRS)

    Samanta, Manoj; Liang, Shoudan; Biegel, Bryan (Technical Monitor)

    2003-01-01

    In today's world, three seemingly diverse fields - computer information technology, nanotechnology and biotechnology are joining forces to enlarge our scientific knowledge and solve complex technological problems. Our group is dedicated to conduct theoretical research exploring the challenges in this area. The major areas of research include: 1) Yeast Protein Interactions; 2) Protein Structures; and 3) Current Transport through Small Molecules.

  12. 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

  13. Manipulating Hydrophobic Interactions in Associative Polymer Solutions via Surfactant-Cyclodextrin Complexation

    NASA Astrophysics Data System (ADS)

    Talwar, Sachin; Harding, Jonathon; Khan, Saad A.

    2008-07-01

    Associative polymers in combination with cyclodextrin (CD) provide a potent tool to manipulate the solution rheology of aqueous solutions. In this study, we discuss the viability and scope of employing surfactants in such systems to facilitate a more versatile and effective tailoring of rheological properties. A model hydrophobically modified alkali-soluble emulsion (HASE) polymer is used which forms a transient physical network of intra- and inter-molecular hydrophobic junctions in solution arising from the interactions between hydrophobic groups grafted on the polymer backbone. The presence of these hydrophobic junctions significantly enhances the solution rheological properties with both the steady state viscosity and dynamic moduli exhibiting an increase by several orders of magnitude. The ability of nonionic surfactants to modulate and recover the hydrophobic interactions in these polymer solutions in the presence of cyclodextrin is examined. The presence of either a- or β-CD results in a dramatic decrease in viscosity and viscoelastic properties of the HASE polymer solution resulting from the encapsulation of polymer hydrophobes by CDs. Addition of nonionic surfactants to such systems promotes a competition between CDs and surfactant molecules to complex with polymer hydrophobes thereby altering the hydrophobic interactions. In this regard, nonylphenol ethoxylates (NPe) with different ethylene oxide (EO) chain lengths, which determine the surfactant hydrophilic-lipophilic balance (HLB), are used.

  14. Novel computational methods to design protein-protein interactions

    NASA Astrophysics Data System (ADS)

    Zhou, Alice Qinhua; O'Hern, Corey; Regan, Lynne

    2014-03-01

    Despite the abundance of structural data, we still cannot accurately predict the structural and energetic changes resulting from mutations at protein interfaces. The inadequacy of current computational approaches to the analysis and design of protein-protein interactions has hampered the development of novel therapeutic and diagnostic agents. In this work, we apply a simple physical model that includes only a minimal set of geometrical constraints, excluded volume, and attractive van der Waals interactions to 1) rank the binding affinity of mutants of tetratricopeptide repeat proteins with their cognate peptides, 2) rank the energetics of binding of small designed proteins to the hydrophobic stem region of the influenza hemagglutinin protein, and 3) predict the stability of T4 lysozyme and staphylococcal nuclease mutants. This work will not only lead to a fundamental understanding of protein-protein interactions, but also to the development of efficient computational methods to rationally design protein interfaces with tunable specificity and affinity, and numerous applications in biomedicine. NSF DMR-1006537, PHY-1019147, Raymond and Beverly Sackler Institute for Biological, Physical and Engineering Sciences, and Howard Hughes Medical Institute.

  15. Systematic computational prediction of protein interaction networks.

    PubMed

    Lees, J G; Heriche, J K; Morilla, I; Ranea, J A; Orengo, C A

    2011-06-01

    Determining the network of physical protein associations is an important first step in developing mechanistic evidence for elucidating biological pathways. Despite rapid advances in the field of high throughput experiments to determine protein interactions, the majority of associations remain unknown. Here we describe computational methods for significantly expanding protein association networks. We describe methods for integrating multiple independent sources of evidence to obtain higher quality predictions and we compare the major publicly available resources available for experimentalists to use. PMID:21572181

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

    PubMed Central

    Khor, Susan

    2014-01-01

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

  17. Features, processing states, and heterologous protein interactions in the modulation of the retroviral nucleocapsid protein function.

    PubMed

    Mirambeau, Gilles; Lyonnais, Sébastien; Gorelick, Robert J

    2010-01-01

    Retroviral nucleocapsid (NC) is central to viral replication. Nucleic acid chaperoning is a key function for NC through the action of its conserved basic amino acids and zinc-finger structures. NC manipulates genomic RNA from its packaging in the producer cell to reverse transcription into the infected host cell. This chaperone function, in conjunction with NC's aggregating properties, is up-modulated by successive NC processing events, from the Gag precursor to the fully mature protein, resulting in the condensation of the nucleocapsid within the capsid shell. Reverse transcription also depends on NC processing, whereas this process provokes NC dissociation from double-stranded DNA, leading to a preintegration complex (PIC), competent for host chromosomal integration. In addition NC interacts with cellular proteins, some of which are involved in viral budding, and also with several viral proteins. All of these properties are reviewed here, focusing on HIV-1 as a paradigmatic reference and highlighting the plasticity of the nucleocapsid architecture. PMID:21045549

  18. Teaching Noncovalent Interactions Using Protein Molecular Evolution

    ERIC Educational Resources Information Center

    Fornasari, Maria Silvina; Parisi, Gustavo; Echave, Julian

    2008-01-01

    Noncovalent interactions and physicochemical properties of amino acids are important topics in biochemistry courses. Here, we present a computational laboratory where the capacity of each of the 20 amino acids to maintain different noncovalent interactions are used to investigate the stabilizing forces in a set of proteins coming from organisms…

  19. Evolving new protein-protein interaction specificity through promiscuous intermediates.

    PubMed

    Aakre, Christopher D; Herrou, Julien; Phung, Tuyen N; Perchuk, Barrett S; Crosson, Sean; Laub, Michael T

    2015-10-22

    Interacting proteins typically coevolve, and the identification of coevolving amino acids can pinpoint residues required for interaction specificity. This approach often assumes that an interface-disrupting mutation in one protein drives selection of a compensatory mutation in its partner during evolution. However, this model requires a non-functional intermediate state prior to the compensatory change. Alternatively, a mutation in one protein could first broaden its specificity, allowing changes in its partner, followed by a specificity-restricting mutation. Using bacterial toxin-antitoxin systems, we demonstrate the plausibility of this second, promiscuity-based model. By screening large libraries of interface mutants, we show that toxins and antitoxins with high specificity are frequently connected in sequence space to more promiscuous variants that can serve as intermediates during a reprogramming of interaction specificity. We propose that the abundance of promiscuous variants promotes the expansion and diversification of toxin-antitoxin systems and other paralogous protein families during evolution. PMID:26478181

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

    PubMed Central

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

    2016-01-01

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

  1. A Protein Interaction Map of Drosophila melanogaster

    NASA Astrophysics Data System (ADS)

    Giot, L.; Bader, J. S.; Brouwer, C.; Chaudhuri, A.; Kuang, B.; Li, Y.; Hao, Y. L.; Ooi, C. E.; Godwin, B.; Vitols, E.; Vijayadamodar, G.; Pochart, P.; Machineni, H.; Welsh, M.; Kong, Y.; Zerhusen, B.; Malcolm, R.; Varrone, Z.; Collis, A.; Minto, M.; Burgess, S.; McDaniel, L.; Stimpson, E.; Spriggs, F.; Williams, J.; Neurath, K.; Ioime, N.; Agee, M.; Voss, E.; Furtak, K.; Renzulli, R.; Aanensen, N.; Carrolla, S.; Bickelhaupt, E.; Lazovatsky, Y.; DaSilva, A.; Zhong, J.; Stanyon, C. A.; Finley, R. L.; White, K. P.; Braverman, M.; Jarvie, T.; Gold, S.; Leach, M.; Knight, J.; Shimkets, R. A.; McKenna, M. P.; Chant, J.; Rothberg, J. M.

    2003-12-01

    Drosophila melanogaster is a proven model system for many aspects of human biology. Here we present a two-hybrid-based protein-interaction map of the fly proteome. A total of 10,623 predicted transcripts were isolated and screened against standard and normalized complementary DNA libraries to produce a draft map of 7048 proteins and 20,405 interactions. A computational method of rating two-hybrid interaction confidence was developed to refine this draft map to a higher confidence map of 4679 proteins and 4780 interactions. Statistical modeling of the network showed two levels of organization: a short-range organization, presumably corresponding to multiprotein complexes, and a more global organization, presumably corresponding to intercomplex connections. The network recapitulated known pathways, extended pathways, and uncovered previously unknown pathway components. This map serves as a starting point for a systems biology modeling of multicellular organisms, including humans.

  2. Computational Methods to Predict Protein Interaction Partners

    NASA Astrophysics Data System (ADS)

    Valencia, Alfonso; Pazos, Florencio

    In the new paradigm for studying biological phenomena represented by Systems Biology, cellular components are not considered in isolation but as forming complex networks of relationships. Protein interaction networks are among the first objects studied from this new point of view. Deciphering the interactome (the whole network of interactions for a given proteome) has been shown to be a very complex task. Computational techniques for detecting protein interactions have become standard tools for dealing with this problem, helping and complementing their experimental counterparts. Most of these techniques use genomic or sequence features intuitively related with protein interactions and are based on "first principles" in the sense that they do not involve training with examples. There are also other computational techniques that use other sources of information (i.e. structural information or even experimental data) or are based on training with examples.

  3. Protein interactions in concentrated ribonuclease solutions

    NASA Astrophysics Data System (ADS)

    Boyer, Mireille; Roy, Marie-Odile; Jullien, Magali; Bonneté, Françoise; Tardieu, Annette

    1999-01-01

    To investigate the protein interactions involved in the crystallization process of ribonuclease A, dynamic light scattering (DLS) and small angle X-ray scattering experiments (SAXS) were performed on concentrated solutions. Whereas the translational diffusion coefficient obtained from DLS is sensitive to thermodynamic and hydrodynamic interactions and permits to calculate an interaction parameter, the shape of the SAXS curves is related to the type of interaction (attractive or repulsive). We compared the effect of pH on protein interactions in the case of two types of crystallizing agents: a mixture of salts (3 M sodium chloride plus 0.2 M ammonium sulfate) and an organic solvent (ethanol). The results show that in the presence of ethanol, as in low salt, protein interactions become more attractive as the pH increases from 4 to 8 and approaches the isoelectric point. In contrast, a reverse effect is observed in high salt conditions: the strength of attractive interactions decreases as the pH increases. The range of the pH effect can be related to ionization of histidine residues, particularly those located in the active site of the protein. The present observations point out the important role played by localized charges in crystallization conditions, whatever the precipitating agent.

  4. Protein-protein interactions and prediction: a comprehensive overview.

    PubMed

    Sowmya, Gopichandran; Ranganathan, Shoba

    2014-01-01

    Molecular function in cellular processes is governed by protein-protein interactions (PPIs) within biological networks. Selective yet specific association of these protein partners contributes to diverse functionality such as catalysis, regulation, assembly, immunity, and inhibition in a cell. Therefore, understanding the principles of protein-protein association has been of immense interest for several decades. We provide an overview of the experimental methods used to determine PPIs and the key databases archiving this information. Structural and functional information of existing protein complexes confers knowledge on the principles of PPI, based on which a classification scheme for PPIs is then introduced. Obtaining high-quality non-redundant datasets of protein complexes for interaction characterisation is an essential step towards deciphering their underlying binding principles. Analysis of physicochemical features and their documentation has enhanced our understanding of the molecular basis of protein-protein association. We describe the diverse datasets created/collected by various groups and their key findings inferring distinguishing features. The currently available interface databases and prediction servers have also been compiled. PMID:23855658

  5. Inferring high-confidence human protein-protein interactions

    PubMed Central

    2012-01-01

    Background As numerous experimental factors drive the acquisition, identification, and interpretation of protein-protein interactions (PPIs), aggregated assemblies of human PPI data invariably contain experiment-dependent noise. Ascertaining the reliability of PPIs collected from these diverse studies and scoring them to infer high-confidence networks is a non-trivial task. Moreover, a large number of PPIs share the same number of reported occurrences, making it impossible to distinguish the reliability of these PPIs and rank-order them. For example, for the data analyzed here, we found that the majority (>83%) of currently available human PPIs have been reported only once. Results In this work, we proposed an unsupervised statistical approach to score a set of diverse, experimentally identified PPIs from nine primary databases to create subsets of high-confidence human PPI networks. We evaluated this ranking method by comparing it with other methods and assessing their ability to retrieve protein associations from a number of diverse and independent reference sets. These reference sets contain known biological data that are either directly or indirectly linked to interactions between proteins. We quantified the average effect of using ranked protein interaction data to retrieve this information and showed that, when compared to randomly ranked interaction data sets, the proposed method created a larger enrichment (~134%) than either ranking based on the hypergeometric test (~109%) or occurrence ranking (~46%). Conclusions From our evaluations, it was clear that ranked interactions were always of value because higher-ranked PPIs had a higher likelihood of retrieving high-confidence experimental data. Reducing the noise inherent in aggregated experimental PPIs via our ranking scheme further increased the accuracy and enrichment of PPIs derived from a number of biologically relevant data sets. These results suggest that using our high-confidence protein interactions

  6. Predicting protein-peptide interactions from scratch

    NASA Astrophysics Data System (ADS)

    Yan, Chengfei; Xu, Xianjin; Zou, Xiaoqin; Zou lab Team

    Protein-peptide interactions play an important role in many cellular processes. The ability to predict protein-peptide complex structures is valuable for mechanistic investigation and therapeutic development. Due to the high flexibility of peptides and lack of templates for homologous modeling, predicting protein-peptide complex structures is extremely challenging. Recently, we have developed a novel docking framework for protein-peptide structure prediction. Specifically, given the sequence of a peptide and a 3D structure of the protein, initial conformations of the peptide are built through protein threading. Then, the peptide is globally and flexibly docked onto the protein using a novel iterative approach. Finally, the sampled modes are scored and ranked by a statistical potential-based energy scoring function that was derived for protein-peptide interactions from statistical mechanics principles. Our docking methodology has been tested on the Peptidb database and compared with other protein-peptide docking methods. Systematic analysis shows significantly improved results compared to the performances of the existing methods. Our method is computationally efficient and suitable for large-scale applications. Nsf CAREER Award 0953839 (XZ) NIH R01GM109980 (XZ).

  7. Topology of Protein Interaction Network Shapes Protein Abundances and Strengths of Their Functional and Nonspecific Interactions

    SciTech Connect

    Maslov, S.; Heo, M.; Shakhnovich, E.

    2011-03-08

    How do living cells achieve sufficient abundances of functional protein complexes while minimizing promiscuous nonfunctional interactions? Here we study this problem using a first-principle model of the cell whose phenotypic traits are directly determined from its genome through biophysical properties of protein structures and binding interactions in a crowded cellular environment. The model cell includes three independent prototypical pathways, whose topologies of protein-protein interaction (PPI) subnetworks are different, but whose contributions to the cell fitness are equal. Model cells evolve through genotypic mutations and phenotypic protein copy number variations. We found a strong relationship between evolved physical-chemical properties of protein interactions and their abundances due to a 'frustration' effect: Strengthening of functional interactions brings about hydrophobic interfaces, which make proteins prone to promiscuous binding. The balancing act is achieved by lowering concentrations of hub proteins while raising solubilities and abundances of functional monomers. On the basis of these principles we generated and analyzed a possible realization of the proteome-wide PPI network in yeast. In this simulation we found that high-throughput affinity capture-mass spectroscopy experiments can detect functional interactions with high fidelity only for high-abundance proteins while missing most interactions for low-abundance proteins.

  8. Protein-protein interactions in reversibly assembled nanopatterns.

    PubMed

    Rakickas, Tomas; Gavutis, Martynas; Reichel, Annett; Piehler, Jacob; Liedberg, Bo; Valiokas, Ramūnas

    2008-10-01

    We describe herein a platform to study protein-protein interactions and to form functional protein complexes in nanoscopic surface domains. For this purpose, we employed multivalent chelator (MCh) templates, which were fabricated in a stepwise procedure combining dip-pen nanolithography (DPN) and molecular recognition-directed assembly. First, we demonstrated that an atomic force microscope (AFM) tip inked with an oligo(ethylene glycol) (OEG) disulfide compound bearing terminal biotin groups can be used to generate biotin patterns on gold achieving line widths below 100 nm, a generic platform for fabrication of functional nanostructures via the highly specific biotin-streptavidin recognition. Subsequently, we converted such biotin/streptavidin patterns into functional MCh patterns for reversible assembly of histidine-tagged (His-tagged) proteins via the attachment of a tris-nitriloacetic acid (trisNTA) biotin derivative. Fluorescence microscopy confirmed reversible immobilization of the receptor subunit ifnar2-His10 and its interaction with interferon-alpha2 labeled with fluorescent quantum dots in a 7 x 7 dot array consisting of trisNTA spots with a diameter of approximately 230 nm. Moreover, we carried out characterization of the specificity, stability, and reversibility as well as quantitative real-time analysis of protein-protein interactions at the fabricated nanopatterns by imaging surface plasmon resonance. Our work offers a route for construction and analysis of functional protein-based nanoarchitectures. PMID:18788824

  9. Predicting protein-protein interactions based only on sequences information.

    PubMed

    Shen, Juwen; Zhang, Jian; Luo, Xiaomin; Zhu, Weiliang; Yu, Kunqian; Chen, Kaixian; Li, Yixue; Jiang, Hualiang

    2007-03-13

    Protein-protein interactions (PPIs) are central to most biological processes. Although efforts have been devoted to the development of methodology for predicting PPIs and protein interaction networks, the application of most existing methods is limited because they need information about protein homology or the interaction marks of the protein partners. In the present work, we propose a method for PPI prediction using only the information of protein sequences. This method was developed based on a learning algorithm-support vector machine combined with a kernel function and a conjoint triad feature for describing amino acids. More than 16,000 diverse PPI pairs were used to construct the universal model. The prediction ability of our approach is better than that of other sequence-based PPI prediction methods because it is able to predict PPI networks. Different types of PPI networks have been effectively mapped with our method, suggesting that, even with only sequence information, this method could be applied to the exploration of networks for any newly discovered protein with unknown biological relativity. In addition, such supplementary experimental information can enhance the prediction ability of the method. PMID:17360525

  10. Building protein interaction maps for Down's syndrome.

    PubMed

    Gardiner, Katheleen; Davisson, Muriel T; Crnic, Linda S

    2004-08-01

    Now that the complete sequences for human chromosome 21 and the orthologous mouse genomic regions are known, reasonably complete, conserved, protein-coding gene catalogues are also available. The central issue now facing Down's syndrome researchers is the correlation of increased expression of specific, normal, chromosome 21 genes with the development of specific deficits in learning and memory. Because of the number of candidate genes involved, the number of alternative splice variants of individual genes and the number of pathways in which these genes function, a pathway analysis approach will be critical to success. Here, three examples, both gene specific and pathway related, that would benefit from pathway analysis are discussed: (1) the potential roles of eight chromosome 21 proteins in RNA processing pathways; (2) the chromosome 21 protein intersectin 1 and its domain composition, alternative splicing, protein interactions and functions; and (3) the interactions of ten chromosome 21 proteins with components of the mitogen-activated protein kinase and the calcineurin signalling pathways. A productive approach to developing gene-phenotype correlations in Down's syndrome will make use of known and predicted functions and interactions of chromosome 21 genes to predict pathways that may be perturbed by their increased levels of expression. Investigations may then be targeted in animal models to specific interactions, intermediate steps or end-points of such pathways and the downstream - perhaps amplified - consequences of gene dosage directly assessed. Once pathway perturbations have been identified, the potential for rational design of therapeutics becomes practical. PMID:15355596

  11. Interaction prediction using conserved network motifs in protein-protein interaction networks

    NASA Astrophysics Data System (ADS)

    Albert, Reka

    2005-03-01

    High-throughput protein interaction detection methods are strongly affected by false positive and false negative results. Focused experiments are needed to complement the large-scale methods by validating previously detected interactions but it is often difficult to decide which proteins to probe as interaction partners. Developing reliable computational methods assisting this decision process is a pressing need in bioinformatics. This talk will describe the recent developments in analyzing and understanding protein interaction networks, then present a method that uses the conserved properties of the protein network to identify and validate interaction candidates. We apply a number of machine learning algorithms to the protein connectivity information and achieve a surprisingly good overall performance in predicting interacting proteins. Using a ``leave-one-ou approach we find average success rates between 20-50% for predicting the correct interaction partner of a protein. We demonstrate that the success of these methods is based on the presence of conserved interaction motifs within the network. A reference implementation and a table with candidate interacting partners for each yeast protein are available at http://www.protsuggest.org

  12. Protein-protein and protein-salt interactions in aqueous protein solutions containing concentrated electrolytes

    SciTech Connect

    Curtis, R.A.; Blanch, H.W.; Prausnitz, J.M.

    1998-01-05

    Protein-protein and protein-salt interactions have been obtained for ovalbumin in solutions of ammonium sulfate and for lysozyme in solutions of ammonium sulfate, sodium chloride, potassium isothiocyanate, and potassium chloride. The two-body interactions between ovalbumin molecules in concentrated ammonium-sulfate solutions can be described by the DLVO potentials plus a potential that accounts for the decrease in free volume available to the protein due to the presence of the salt ions. The interaction between ovalbumin and ammonium sulfate is unfavorable, reflecting the kosmotropic nature of sulfate anions. Lysozyme-lysozyme interactions cannot be described by the above potentials because anion binding to lysozyme alters these interactions. Lysozyme-isothiocyanate complexes are strongly attractive due to electrostatic interactions resulting from bridging by the isothiocyanate ion. Lysozyme-lysozyme interactions in sulfate solutions are more repulsive than expected, possibly resulting from a larger excluded volume of a lysozyme-sulfate bound complex or perhaps, hydration forces between the lysozyme-sulfate complexes.

  13. Effects of Dispositional Ability Conceptions, Manipulated Learning Environments, and Intrinsic Motivation on Persistence and Performance: An Interaction Approach

    ERIC Educational Resources Information Center

    Li, Weidong; Lee, Amelia M.; Solmon, Melinda

    2008-01-01

    The present study used an interaction approach to investigate how individuals' dispositions about ability as incremental or fixed (entity), manipulated learning environments, and intrinsic motivation affect persistence and performance on a challenging, novel motor skill. Seventy-two female college students who were assigned to either an…

  14. [Chemical libraries dedicated to protein-protein interactions].

    PubMed

    Sperandio, Olivier; Villoutreix, Bruno O; Morelli, Xavier; Roche, Philippe

    2015-03-01

    The identification of complete networks of protein-protein interactions (PPI) within a cell has contributed to major breakthroughs in understanding biological pathways, host-pathogen interactions and cancer development. As a consequence, PPI have emerged as a new class of promising therapeutic targets. However, they are still considered as a challenging class of targets for drug discovery programs. Recent successes have allowed the characterization of structural and physicochemical properties of protein-protein interfaces leading to a better understanding of how they can be disrupted with small molecule compounds. In addition, characterization of the profiles of PPI inhibitors has allowed the development of PPI-focused libraries. In this review, we present the current efforts at developing chemical libraries dedicated to these innovative targets. PMID:25855285

  15. KFC Server: interactive forecasting of protein interaction hot spots.

    PubMed

    Darnell, Steven J; LeGault, Laura; Mitchell, Julie C

    2008-07-01

    The KFC Server is a web-based implementation of the KFC (Knowledge-based FADE and Contacts) model-a machine learning approach for the prediction of binding hot spots, or the subset of residues that account for most of a protein interface's; binding free energy. The server facilitates the automated analysis of a user submitted protein-protein or protein-DNA interface and the visualization of its hot spot predictions. For each residue in the interface, the KFC Server characterizes its local structural environment, compares that environment to the environments of experimentally determined hot spots and predicts if the interface residue is a hot spot. After the computational analysis, the user can visualize the results using an interactive job viewer able to quickly highlight predicted hot spots and surrounding structural features within the protein structure. The KFC Server is accessible at http://kfc.mitchell-lab.org. PMID:18539611

  16. Prediction of protein-protein interactions based on protein-protein correlation using least squares regression.

    PubMed

    Huang, De-Shuang; Zhang, Lei; Han, Kyungsook; Deng, Suping; Yang, Kai; Zhang, Hongbo

    2014-01-01

    In order to transform protein sequences into the feature vectors, several works have been done, such as computing auto covariance (AC), conjoint triad (CT), local descriptor (LD), moran autocorrelation (MA), normalized moreaubroto autocorrelation (NMB) and so on. In this paper, we shall adopt these transformation methods to encode the proteins, respectively, where AC, CT, LD, MA and NMB are all represented by '+' in a unified manner. A new method, i.e. the combination of least squares regression with '+' (abbreviated as LSR(+)), will be introduced for encoding a protein-protein correlation-based feature representation and an interacting protein pair. Thus there are totally five different combinations for LSR(+), i.e. LSRAC, LSRCT, LSRLD, LSRMA and LSRNMB. As a result, we combined a support vector machine (SVM) approach with LSR(+) to predict protein-protein interactions (PPI) and PPI networks. The proposed method has been applied on four datasets, i.e. Saaccharomyces cerevisiae, Escherichia coli, Homo sapiens and Caenorhabditis elegans. The experimental results demonstrate that all LSR(+) methods outperform many existing representative algorithms. Therefore, LSR(+) is a powerful tool to characterize the protein-protein correlations and to infer PPI, whilst keeping high performance on prediction of PPI networks. PMID:25059329

  17. Annotation and retrieval in protein interaction databases

    NASA Astrophysics Data System (ADS)

    Cannataro, Mario; Hiram Guzzi, Pietro; Veltri, Pierangelo

    2014-06-01

    Biological databases have been developed with a special focus on the efficient retrieval of single records or the efficient computation of specialized bioinformatics algorithms against the overall database, such as in sequence alignment. The continuos production of biological knowledge spread on several biological databases and ontologies, such as Gene Ontology, and the availability of efficient techniques to handle such knowledge, such as annotation and semantic similarity measures, enable the development on novel bioinformatics applications that explicitly use and integrate such knowledge. After introducing the annotation process and the main semantic similarity measures, this paper shows how annotations and semantic similarity can be exploited to improve the extraction and analysis of biologically relevant data from protein interaction databases. As case studies, the paper presents two novel software tools, OntoPIN and CytoSeVis, both based on the use of Gene Ontology annotations, for the advanced querying of protein interaction databases and for the enhanced visualization of protein interaction networks.

  18. Transient DNA / RNA-protein interactions.

    PubMed

    Blanco, Francisco J; Montoya, Guillermo

    2011-05-01

    The great pace of biomolecular structure determination has provided a plethora of protein structures, but not as many structures of nucleic acids or of their complexes with proteins. The recognition of DNA and RNA molecules by proteins may produce large and relatively stable assemblies (such as the ribosome) or transient complexes (such as DNA clamps sliding through the DNA). These transient interactions are most difficult to characterize, but even in 'stable' complexes captured in crystal structures, the dynamics of the whole or part of the assembly pose great technical difficulties in understanding their function. The development and refinement of powerful experimental and computational tools have made it possible to learn a great deal about the relevance of these fleeting events for numerous biological processes. We discuss here the most recent findings and the challenges that lie ahead in the quest for a better understanding of protein-nucleic acid interactions. PMID:21410646

  19. Studying protein-protein interactions: progress, pitfalls and solutions.

    PubMed

    Hayes, Sheri; Malacrida, Beatrice; Kiely, Maeve; Kiely, Patrick A

    2016-08-15

    Signalling proteins are intrinsic to all biological processes and interact with each other in tightly regulated and orchestrated signalling complexes and pathways. Characterization of protein binding can help to elucidate protein function within signalling pathways. This information is vital for researchers to gain a more comprehensive knowledge of cellular networks which can then be used to develop new therapeutic strategies for disease. However, studying protein-protein interactions (PPIs) can be challenging as the interactions can be extremely transient downstream of specific environmental cues. There are many powerful techniques currently available to identify and confirm PPIs. Choosing the most appropriate range of techniques merits serious consideration. The aim of this review is to provide a starting point for researchers embarking on a PPI study. We provide an overview and point of reference for some of the many methods available to identify interactions from in silico analysis and large scale screening tools through to the methods used to validate potential PPIs. We discuss the advantages and disadvantages of each method and we also provide a workflow chart to highlight the main experimental questions to consider when planning cell lysis to maximize experimental success. PMID:27528744

  20. Potential disruption of protein-protein interactions by graphene oxide.

    PubMed

    Feng, Mei; Kang, Hongsuk; Yang, Zaixing; Luan, Binquan; Zhou, Ruhong

    2016-06-14

    Graphene oxide (GO) is a promising novel nanomaterial with a wide range of potential biomedical applications due to its many intriguing properties. However, very little research has been conducted to study its possible adverse effects on protein-protein interactions (and thus subsequent toxicity to human). Here, the potential cytotoxicity of GO is investigated at molecular level using large-scale, all-atom molecular dynamics simulations to explore the interaction mechanism between a protein dimer and a GO nanosheet oxidized at different levels. Our theoretical results reveal that GO nanosheet could intercalate between the two monomers of HIV-1 integrase dimer, disrupting the protein-protein interactions and eventually lead to dimer disassociation as graphene does [B. Luan et al., ACS Nano 9(1), 663 (2015)], albeit its insertion process is slower when compared with graphene due to the additional steric and attractive interactions. This study helps to better understand the toxicity of GO to cell functions which could shed light on how to improve its biocompatibility and biosafety for its wide potential biomedical applications. PMID:27306022

  1. Potential disruption of protein-protein interactions by graphene oxide

    NASA Astrophysics Data System (ADS)

    Feng, Mei; Kang, Hongsuk; Yang, Zaixing; Luan, Binquan; Zhou, Ruhong

    2016-06-01

    Graphene oxide (GO) is a promising novel nanomaterial with a wide range of potential biomedical applications due to its many intriguing properties. However, very little research has been conducted to study its possible adverse effects on protein-protein interactions (and thus subsequent toxicity to human). Here, the potential cytotoxicity of GO is investigated at molecular level using large-scale, all-atom molecular dynamics simulations to explore the interaction mechanism between a protein dimer and a GO nanosheet oxidized at different levels. Our theoretical results reveal that GO nanosheet could intercalate between the two monomers of HIV-1 integrase dimer, disrupting the protein-protein interactions and eventually lead to dimer disassociation as graphene does [B. Luan et al., ACS Nano 9(1), 663 (2015)], albeit its insertion process is slower when compared with graphene due to the additional steric and attractive interactions. This study helps to better understand the toxicity of GO to cell functions which could shed light on how to improve its biocompatibility and biosafety for its wide potential biomedical applications.

  2. Peptiderive server: derive peptide inhibitors from protein-protein interactions.

    PubMed

    Sedan, Yuval; Marcu, Orly; Lyskov, Sergey; Schueler-Furman, Ora

    2016-07-01

    The Rosetta Peptiderive protocol identifies, in a given structure of a protein-protein interaction, the linear polypeptide segment suggested to contribute most to binding energy. Interactions that feature a 'hot segment', a linear peptide with significant binding energy compared to that of the complex, may be amenable for inhibition and the peptide sequence and structure derived from the interaction provide a starting point for rational drug design. Here we present a web server for Peptiderive, which is incorporated within the ROSIE web interface for Rosetta protocols. A new feature of the protocol also evaluates whether derived peptides are good candidates for cyclization. Fast computation times and clear visualization allow users to quickly assess the interaction of interest. The Peptiderive server is available for free use at http://rosie.rosettacommons.org/peptiderive. PMID:27141963

  3. A Method for Predicting Protein-Protein Interaction Types

    PubMed Central

    Silberberg, Yael

    2014-01-01

    Protein-protein interactions (PPIs) govern basic cellular processes through signal transduction and complex formation. The diversity of those processes gives rise to a remarkable diversity of interactions types, ranging from transient phosphorylation interactions to stable covalent bonding. Despite our increasing knowledge on PPIs in humans and other species, their types remain relatively unexplored and few annotations of types exist in public databases. Here, we propose the first method for systematic prediction of PPI type based solely on the techniques by which the interaction was detected. We show that different detection methods are better suited for detecting specific types. We apply our method to ten interaction types on a large scale human PPI dataset. We evaluate the performance of the method using both internal cross validation and external data sources. In cross validation, we obtain an area under receiver operating characteristic (ROC) curve ranging from 0.65 to 0.97 with an average of 0.84 across the predicted types. Comparing the predicted interaction types to external data sources, we obtained significant agreements for phosphorylation and ubiquitination interactions, with hypergeometric p-value = 2.3e−54 and 5.6e−28 respectively. We examine the biological relevance of our predictions using known signaling pathways and chart the abundance of interaction types in cell processes. Finally, we investigate the cross-relations between different interaction types within the network and characterize the discovered patterns, or motifs. We expect the resulting annotated network to facilitate the reconstruction of process-specific subnetworks and assist in predicting protein function or interaction. PMID:24625764

  4. Targeting Protein-Protein Interactions for Parasite Control

    PubMed Central

    Taylor, Christina M.; Fischer, Kerstin; Abubucker, Sahar; Wang, Zhengyuan; Martin, John; Jiang, Daojun; Magliano, Marc; Rosso, Marie-Noëlle; Li, Ben-Wen; Fischer, Peter U.; Mitreva, Makedonka

    2011-01-01

    Finding new drug targets for pathogenic infections would be of great utility for humanity, as there is a large need to develop new drugs to fight infections due to the developing resistance and side effects of current treatments. Current drug targets for pathogen infections involve only a single protein. However, proteins rarely act in isolation, and the majority of biological processes occur via interactions with other proteins, so protein-protein interactions (PPIs) offer a realm of unexplored potential drug targets and are thought to be the next-generation of drug targets. Parasitic worms were chosen for this study because they have deleterious effects on human health, livestock, and plants, costing society billions of dollars annually and many sequenced genomes are available. In this study, we present a computational approach that utilizes whole genomes of 6 parasitic and 1 free-living worm species and 2 hosts. The species were placed in orthologous groups, then binned in species-specific ortholgous groups. Proteins that are essential and conserved among species that span a phyla are of greatest value, as they provide foundations for developing broad-control strategies. Two PPI databases were used to find PPIs within the species specific bins. PPIs with unique helminth proteins and helminth proteins with unique features relative to the host, such as indels, were prioritized as drug targets. The PPIs were scored based on RNAi phenotype and homology to the PDB (Protein DataBank). EST data for the various life stages, GO annotation, and druggability were also taken into consideration. Several PPIs emerged from this study as potential drug targets. A few interactions were supported by co-localization of expression in M. incognita (plant parasite) and B. malayi (H. sapiens parasite), which have extremely different modes of parasitism. As more genomes of pathogens are sequenced and PPI databases expanded, this methodology will become increasingly applicable. PMID

  5. Manipulations of amyloid precursor protein cleavage disrupt the circadian clock in aging Drosophila.

    PubMed

    Blake, Matthew R; Holbrook, Scott D; Kotwica-Rolinska, Joanna; Chow, Eileen S; Kretzschmar, Doris; Giebultowicz, Jadwiga M

    2015-05-01

    Alzheimer's disease (AD) is a neurodegenerative disease characterized by severe cognitive deterioration. While causes of AD pathology are debated, a large body of evidence suggests that increased cleavage of Amyloid Precursor Protein (APP) producing the neurotoxic Amyloid-β (Aβ) peptide plays a fundamental role in AD pathogenesis. One of the detrimental behavioral symptoms commonly associated with AD is the fragmentation of sleep-activity cycles with increased nighttime activity and daytime naps in humans. Sleep-activity cycles, as well as physiological and cellular rhythms, which may be important for neuronal homeostasis, are generated by a molecular system known as the circadian clock. Links between AD and the circadian system are increasingly evident but not well understood. Here we examined whether genetic manipulations of APP-like (APPL) protein cleavage in Drosophila melanogaster affect rest-activity rhythms and core circadian clock function in this model organism. We show that the increased β-cleavage of endogenous APPL by the β-secretase (dBACE) severely disrupts circadian behavior and leads to reduced expression of clock protein PER in central clock neurons of aging flies. Our data suggest that behavioral rhythm disruption is not a product of APPL-derived Aβ production but rather may be caused by a mechanism common to both α and β-cleavage pathways. Specifically, we show that increased production of the endogenous Drosophila Amyloid Intracellular Domain (dAICD) caused disruption of circadian rest-activity rhythms, while flies overexpressing endogenous APPL maintained stronger circadian rhythms during aging. In summary, our study offers a novel entry point toward understanding the mechanism of circadian rhythm disruption in Alzheimer's disease. PMID:25766673

  6. Targeting Protein–Protein Interactions in the HIF System

    PubMed Central

    Abboud, Martine I.; Hancock, Rebecca L.

    2016-01-01

    Abstract Animals respond to chronic hypoxia by increasing the levels of a transcription factor known as the hypoxia‐inducible factor (HIF). HIF upregulates multiple genes, the products of which work to ameliorate the effects of limited oxygen at cellular and systemic levels. Hypoxia sensing by the HIF system involves hydroxylase‐catalysed post‐translational modifications of the HIF α‐subunits, which 1) signal for degradation of HIF‐α and 2) limit binding of HIF to transcriptional coactivator proteins. Because the hypoxic response is relevant to multiple disease states, therapeutic manipulation of the HIF‐mediated response has considerable medicinal potential. In addition to modulation of catalysis by the HIF hydroxylases, the HIF system manifests other possibilities for therapeutic intervention involving protein–protein and protein–nucleic acid interactions. Recent advances in our understanding of the structural biology and biochemistry of the HIF system are facilitating medicinal chemistry efforts. Herein we give an overview of the HIF system, focusing on structural knowledge of protein–protein interactions and how this might be used to modulate the hypoxic response for therapeutic benefit. PMID:26997519

  7. Learning compliant manipulation through kinesthetic and tactile human-robot interaction.

    PubMed

    Kronander, Klas; Billard, Aude

    2014-01-01

    Robot Learning from Demonstration (RLfD) has been identified as a key element for making robots useful in daily lives. A wide range of techniques has been proposed for deriving a task model from a set of demonstrations of the task. Most previous works use learning to model the kinematics of the task, and for autonomous execution the robot then relies on a stiff position controller. While many tasks can and have been learned this way, there are tasks in which controlling the position alone is insufficient to achieve the goals of the task. These are typically tasks that involve contact or require a specific response to physical perturbations. The question of how to adjust the compliance to suit the need of the task has not yet been fully treated in Robot Learning from Demonstration. In this paper, we address this issue and present interfaces that allow a human teacher to indicate compliance variations by physically interacting with the robot during task execution. We validate our approach in two different experiments on the 7 DoF Barrett WAM and KUKA LWR robot manipulators. Furthermore, we conduct a user study to evaluate the usability of our approach from a non-roboticists perspective. PMID:25248219

  8. Quantitative study of protein-protein interactions by quartz nanopipettes

    NASA Astrophysics Data System (ADS)

    Tiwari, Purushottam Babu; Astudillo, Luisana; Miksovska, Jaroslava; Wang, Xuewen; Li, Wenzhi; Darici, Yesim; He, Jin

    2014-08-01

    In this report, protein-modified quartz nanopipettes were used to quantitatively study protein-protein interactions in attoliter sensing volumes. As shown by numerical simulations, the ionic current through the conical-shaped nanopipette is very sensitive to the surface charge variation near the pore mouth. With the appropriate modification of negatively charged human neuroglobin (hNgb) onto the inner surface of a nanopipette, we were able to detect concentration-dependent current change when the hNgb-modified nanopipette tip was exposed to positively charged cytochrome c (Cyt c) with a series of concentrations in the bath solution. Such current change is due to the adsorption of Cyt c to the inner surface of the nanopipette through specific interactions with hNgb. In contrast, a smaller current change with weak concentration dependence was observed when Cyt c was replaced with lysozyme, which does not specifically bind to hNgb. The equilibrium dissociation constant (KD) for the Cyt c-hNgb complex formation was derived and the value matched very well with the result from surface plasmon resonance measurement. This is the first quantitative study of protein-protein interactions by a conical-shaped nanopore based on charge sensing. Our results demonstrate that nanopipettes can potentially be used as a label-free analytical tool to quantitatively characterize protein-protein interactions.In this report, protein-modified quartz nanopipettes were used to quantitatively study protein-protein interactions in attoliter sensing volumes. As shown by numerical simulations, the ionic current through the conical-shaped nanopipette is very sensitive to the surface charge variation near the pore mouth. With the appropriate modification of negatively charged human neuroglobin (hNgb) onto the inner surface of a nanopipette, we were able to detect concentration-dependent current change when the hNgb-modified nanopipette tip was exposed to positively charged cytochrome c (Cyt c) with

  9. Experimental evolution of protein–protein interaction networks

    PubMed Central

    Kaçar, Betül; Gaucher, Eric A.

    2013-01-01

    The modern synthesis of evolutionary theory and genetics has enabled us to discover underlying molecular mechanisms of organismal evolution. We know that in order to maximize an organism's fitness in a particular environment, individual interactions among components of protein and nucleic acid networks need to be optimized by natural selection, or sometimes through random processes, as the organism responds to changes and/or challenges in the environment. Despite the significant role of molecular networks in determining an organism's adaptation to its environment, we still do not know how such inter- and intra-molecular interactions within networks change over time and contribute to an organism's evolvability while maintaining overall network functions. One way to address this challenge is to identify connections between molecular networks and their host organisms, to manipulate these connections, and then attempt to understand how such perturbations influence molecular dynamics of the network and thus influence evolutionary paths and organismal fitness. In the present review, we discuss how integrating evolutionary history with experimental systems that combine tools drawn from molecular evolution, synthetic biology and biochemistry allow us to identify the underlying mechanisms of organismal evolution, particularly from the perspective of protein interaction networks. PMID:23849056

  10. Prediction and redesign of protein-protein interactions.

    PubMed

    Lua, Rhonald C; Marciano, David C; Katsonis, Panagiotis; Adikesavan, Anbu K; Wilkins, Angela D; Lichtarge, Olivier

    2014-01-01

    Understanding the molecular basis of protein function remains a central goal of biology, with the hope to elucidate the role of human genes in health and in disease, and to rationally design therapies through targeted molecular perturbations. We review here some of the computational techniques and resources available for characterizing a critical aspect of protein function - those mediated by protein-protein interactions (PPI). We describe several applications and recent successes of the Evolutionary Trace (ET) in identifying molecular events and shapes that underlie protein function and specificity in both eukaryotes and prokaryotes. ET is a part of analytical approaches based on the successes and failures of evolution that enable the rational control of PPI. PMID:24878423

  11. The Arabidopsis ESCRT protein-protein interaction network.

    PubMed

    Shahriari, Mojgan; Richter, Klaus; Keshavaiah, Channa; Sabovljevic, Aneta; Huelskamp, Martin; Schellmann, Swen

    2011-05-01

    In yeast, endosomal sorting of monoubiquitylated transmembrane proteins is performed by a subset of the 19 "class E vacuolar protein sorting" proteins. The core machinery consists of 11 proteins that are organised in three complexes termed ESCRT I-III (endosomal sorting complex required for transport I-III) and is conserved in eukaryotic cells. While the pathway is well understood in yeast and animals, the plant ESCRT system is largely unexplored. At least one sequence homolog for each ESCRT component can be found in the Arabidopsis genome. Generally, sequence conservation between yeast/animals and the Arabidopsis proteins is low. To understand details about participating proteins and complex organization we have performed a systematic pairwise yeast two hybrid analysis of all Arabidopsis proteins showing homology to the ESCRT core machinery. Positive interactions were validated using bimolecular fluorescence complementation. In our experiments, most putative ESCRT components exhibited interactions with other ESCRT components that could be shown to occur on endosomes suggesting that despite their low homology to their yeast and animal counterparts they represent functional components of the plant ESCRT pathway. PMID:21442383

  12. Functional manipulation of a calcium-binding protein from Entamoeba histolytica guided by paramagnetic NMR.

    PubMed

    Rout, Ashok K; Patel, Sunita; Somlata; Shukla, Manish; Saraswathi, Deepa; Bhattacharya, Alok; Chary, Kandala V R

    2013-08-01

    EhCaBP1, one of the calcium-binding proteins from Entamoeba histolytica, is a two-domain EF-hand protein. The two domains of EhCaBP1 are structurally and functionally different from each other. However, both domains are required for structural stability and a full range of functional diversity. Analysis of sequence and structure of EhCaBP1 and other CaBPs indicates that the C-terminal domain of EhCaBP1 possesses a unique structure compared with other family members. This had been attributed to the absence of a Phe-Phe interaction between highly conserved Phe residues at the -4 position in EF-hand III (F[-4]; Tyr(81)) and at the 13th position in EF-hand IV (F[+13]; Phe(129)) of the C-terminal domain. Against this backdrop, we mutated the Tyr residue at the -4th position of EF III to the Phe residue (Y81F), to bring in the Phe-Phe interaction and understand the nature of structural and functional changes in the protein by NMR spectroscopy, molecular dynamics (MD) simulation, isothermal titration calorimetry (ITC), and biological assays, such as imaging and actin binding. The Y81F mutation in EhCaBP1 resulted in a more compact structure for the C-terminal domain of the mutant as in the case of calmodulin and troponin C. The compact structure is favored by the presence of a π-π interaction between Phe(81) and Phe(129) along with several hydrophobic interactions of Phe(81), which are not seen in the wild-type protein. Furthermore, the biological assays reveal preferential membrane localization of the mutant, loss of its colocalization with actin in the phagocytic cups, whereas retaining its ability to bind G- and F-actin. PMID:23782698

  13. Manipulation of the host protein acetylation network by human immunodeficiency virus type 1

    PubMed Central

    Jeng, Mark Y.; Ali, Ibraheem; Ott, Melanie

    2016-01-01

    Over the last 15 years, protein acetylation has emerged as a globally important post-translational modification that fine-tunes major cellular processes in many life forms. This dynamic regulatory system is critical both for complex eukaryotic cells and for the viruses that infect them. HIV-1 accesses the host acetylation network by interacting with several key enzymes, thereby promoting infection at multiple steps during the viral life cycle. Inhibitors of host histone deacetylases and bromodomain-containing proteins are now being pursued as therapeutic strategies to enhance current antiretroviral treatment. As more acetylation-targeting compounds are reaching clinical trials, it is timely to review the role of reversible protein acetylation in HIV-infected CD4+ T cells. PMID:26329395

  14. Self diffusion of interacting membrane proteins.

    PubMed Central

    Abney, J R; Scalettar, B A; Owicki, J C

    1989-01-01

    A two-dimensional version of the generalized Smoluchowski equation is used to analyze the time (or distance) dependent self diffusion of interacting membrane proteins in concentrated membrane systems. This equation provides a well established starting point for descriptions of the diffusion of particles that interact through both direct and hydrodynamic forces; in this initial work only the effects of direct interactions are explicitly considered. Data describing diffusion in the presence of hard-core repulsions, soft repulsions, and soft repulsions with weak attractions are presented. The effect that interactions have on the self-diffusion coefficient of a real protein molecule from mouse liver gap junctions is also calculated. The results indicate that self diffusion is always inhibited by direct interactions; this observation is interpreted in terms of the caging that will exist at finite protein concentration. It is also noted that, over small distance scales, the diffusion coefficient is determined entirely by the very strong Brownian forces; therefore, as a function of displacement the self-diffusion coefficient decays (rapidly) from its value at infinite dilution to its steady-state interaction-averaged value. The steady-state self-diffusion coefficient describes motion over distance scales that range from approximately 10 nm to cellular dimensions and is the quantity measured in fluorescence recovery after photobleaching experiments. The short-ranged behavior of the diffusion coefficient is important on the interparticle-distance scale and may therefore influence the rate at which nearest-neighbor collisional processes take place. The hard-disk theoretical results presented here are in excellent agreement with lattice Monte-Carlo results obtained by other workers. The concentration dependence of experimentally measured diffusion coefficients of antibody-hapten complexes bound to the membrane surface is consistent with that predicted by the theory. The

  15. Dynamic interactions of proteins in complex networks

    SciTech Connect

    Appella, E.; Anderson, C.

    2009-10-01

    Recent advances in techniques such as NMR and EPR spectroscopy have enabled the elucidation of how proteins undergo structural changes to act in concert in complex networks. The three minireviews in this series highlight current findings and the capabilities of new methodologies for unraveling the dynamic changes controlling diverse cellular functions. They represent a sampling of the cutting-edge research presented at the 17th Meeting of Methods in Protein Structure Analysis, MPSA2008, in Sapporo, Japan, 26-29 August, 2008 (http://www.iapsap.bnl.gov). The first minireview, by Christensen and Klevit, reports on a structure-based yeast two-hybrid method for identifying E2 ubiquitin-conjugating enzymes that interact with the E3 BRCA1/BARD1 heterodimer ligase to generate either mono- or polyubiquitinated products. This method demonstrated for the first time that the BRCA1/BARD1 E3 can interact with 10 different E2 enzymes. Interestingly, the interaction with multiple E2 enzymes displayed unique ubiquitin-transfer properties, a feature expected to be common among other RING and U-box E3s. Further characterization of new E3 ligases and the E2 enzymes that interact with them will greatly enhance our understanding of ubiquitin transfer and facilitate studies of roles of ubiquitin and ubiquitin-like proteins in protein processing and trafficking. Stein et al., in the second minireview, describe recent progress in defining the binding specificity of different peptide-binding domains. The authors clearly point out that transient peptide interactions mediated by both post-translational modifications and disordered regions ensure a high level of specificity. They postulate that a regulatory code may dictate the number of combinations of domains and post-translational modifications needed to achieve the required level of interaction specificity. Moreover, recognition alone is not enough to obtain a stable complex, especially in a complex cellular environment. Increasing

  16. Motif mediated protein-protein interactions as drug targets.

    PubMed

    Corbi-Verge, Carles; Kim, Philip M

    2016-01-01

    Protein-protein interactions (PPI) are involved in virtually every cellular process and thus represent an attractive target for therapeutic interventions. A significant number of protein interactions are frequently formed between globular domains and short linear peptide motifs (DMI). Targeting these DMIs has proven challenging and classical approaches to inhibiting such interactions with small molecules have had limited success. However, recent new approaches have led to the discovery of potent inhibitors, some of them, such as Obatoclax, ABT-199, AEG-40826 and SAH-p53-8 are likely to become approved drugs. These novel inhibitors belong to a wide range of different molecule classes, ranging from small molecules to peptidomimetics and biologicals. This article reviews the main reasons for limited success in targeting PPIs, discusses how successful approaches overcome these obstacles to discovery promising inhibitors for human protein double minute 2 (HDM2), B-cell lymphoma 2 (Bcl-2), X-linked inhibitor of apoptosis protein (XIAP), and provides a summary of the promising approaches currently in development that indicate the future potential of PPI inhibitors in drug discovery. PMID:26936767

  17. Modulation of opioid receptor function by protein-protein interactions.

    PubMed

    Alfaras-Melainis, Konstantinos; Gomes, Ivone; Rozenfeld, Raphael; Zachariou, Venetia; Devi, Lakshmi

    2009-01-01

    Opioid receptors, MORP, DORP and KORP, belong to the family A of G protein coupled receptors (GPCR), and have been found to modulate a large number of physiological functions, including mood, stress, appetite, nociception and immune responses. Exogenously applied opioid alkaloids produce analgesia, hedonia and addiction. Addiction is linked to alterations in function and responsiveness of all three opioid receptors in the brain. Over the last few years, a large number of studies identified protein-protein interactions that play an essential role in opioid receptor function and responsiveness. Here, we summarize interactions shown to affect receptor biogenesis and trafficking, as well as those affecting signal transduction events following receptor activation. This article also examines protein interactions modulating the rate of receptor endocytosis and degradation, events that play a major role in opiate analgesia. Like several other GPCRs, opioid receptors may form homo or heterodimers. The last part of this review summarizes recent knowledge on proteins known to affect opioid receptor dimerization. PMID:19273296

  18. Docking and scoring protein interactions: CAPRI 2009.

    PubMed

    Lensink, Marc F; Wodak, Shoshana J

    2010-11-15

    Protein docking algorithms are assessed by evaluating blind predictions performed during 2007-2009 in Rounds 13-19 of the community-wide experiment on critical assessment of predicted interactions (CAPRI). We evaluated the ability of these algorithms to sample docking poses and to single out specific association modes in 14 targets, representing 11 distinct protein complexes. These complexes play important biological roles in RNA maturation, G-protein signal processing, and enzyme inhibition and function. One target involved protein-RNA interactions not previously considered in CAPRI, several others were hetero-oligomers, or featured multiple interfaces between the same protein pair. For most targets, predictions started from the experimentally determined structures of the free (unbound) components, or from models built from known structures of related or similar proteins. To succeed they therefore needed to account for conformational changes and model inaccuracies. In total, 64 groups and 12 web-servers submitted docking predictions of which 4420 were evaluated. Overall our assessment reveals that 67% of the groups, more than ever before, produced acceptable models or better for at least one target, with many groups submitting multiple high- and medium-accuracy models for two to six targets. Forty-one groups including four web-servers participated in the scoring experiment with 1296 evaluated models. Scoring predictions also show signs of progress evidenced from the large proportion of correct models submitted. But singling out the best models remains a challenge, which also adversely affects the ability to correctly rank docking models. With the increased interest in translating abstract protein interaction networks into realistic models of protein assemblies, the growing CAPRI community is actively developing more efficient and reliable docking and scoring methods for everyone to use. PMID:20806235

  19. RAID: a comprehensive resource for human RNA-associated (RNA-RNA/RNA-protein) interaction.

    PubMed

    Zhang, Xiaomeng; Wu, Deng; Chen, Liqun; Li, Xiang; Yang, Jinxurong; Fan, Dandan; Dong, Tingting; Liu, Mingyue; Tan, Puwen; Xu, Jintian; Yi, Ying; Wang, Yuting; Zou, Hua; Hu, Yongfei; Fan, Kaili; Kang, Juanjuan; Huang, Yan; Miao, Zhengqiang; Bi, Miaoman; Jin, Nana; Li, Kongning; Li, Xia; Xu, Jianzhen; Wang, Dong

    2014-07-01

    Transcriptomic analyses have revealed an unexpected complexity in the eukaryote transcriptome, which includes not only protein-coding transcripts but also an expanding catalog of noncoding RNAs (ncRNAs). Diverse coding and noncoding RNAs (ncRNAs) perform functions through interaction with each other in various cellular processes. In this project, we have developed RAID (http://www.rna-society.org/raid), an RNA-associated (RNA-RNA/RNA-protein) interaction database. RAID intends to provide the scientific community with all-in-one resources for efficient browsing and extraction of the RNA-associated interactions in human. This version of RAID contains more than 6100 RNA-associated interactions obtained by manually reviewing more than 2100 published papers, including 4493 RNA-RNA interactions and 1619 RNA-protein interactions. Each entry contains detailed information on an RNA-associated interaction, including RAID ID, RNA/protein symbol, RNA/protein categories, validated method, expressing tissue, literature references (Pubmed IDs), and detailed functional description. Users can query, browse, analyze, and manipulate RNA-associated (RNA-RNA/RNA-protein) interaction. RAID provides a comprehensive resource of human RNA-associated (RNA-RNA/RNA-protein) interaction network. Furthermore, this resource will help in uncovering the generic organizing principles of cellular function network. PMID:24803509

  20. Dynamic regulation of lipid-protein interactions.

    PubMed

    Martfeld, Ashley N; Rajagopalan, Venkatesan; Greathouse, Denise V; Koeppe, Roger E

    2015-09-01

    We review the importance of helix motions for the function of several important categories of membrane proteins and for the properties of several model molecular systems. For voltage-gated potassium or sodium channels, sliding, tilting and/or rotational movements of the S4 helix accompanied by a swapping of cognate side-chain ion-pair interactions regulate the channel gating. In the seven-helix G protein-coupled receptors, exemplified by the rhodopsins, collective helix motions serve to activate the functional signaling. Peptides which initially associate with lipid-bilayer membrane surfaces may undergo dynamic transitions from surface-bound to tilted-transmembrane orientations, sometimes accompanied by changes in the molecularity, formation of a pore or, more generally, the activation of biological function. For single-span membrane proteins, such as the tyrosine kinases, an interplay between juxtamembrane and transmembrane domains is likely to be crucial for the regulation of dimer assembly that in turn is associated with the functional responses to external signals. Additionally, we note that experiments with designed single-span transmembrane helices offer fundamental insights into the molecular features that govern protein-lipid interactions. This article is part of a Special Issue entitled: Lipid-protein interactions. PMID:25666872

  1. Applications of the Restriction Free (RF) cloning procedure for molecular manipulations and protein expression.

    PubMed

    Unger, Tamar; Jacobovitch, Yossi; Dantes, Ada; Bernheim, Reut; Peleg, Yoav

    2010-10-01

    Molecular manipulations, including DNA cloning and mutagenesis are basic tools used on a routine basis in all life-science disciplines. Over the last decade new methodologies have emerged that facilitated and expanded the applications for DNA cloning and mutagenesis. Ligation-Independent Cloning (LIC) techniques were developed and replaced the classical Ligation Dependent Cloning (LDC) platform. Restriction Free (RF) cloning was originally developed for introduction of foreign DNA into a plasmid at any predetermined position. RF cloning is based on PCR amplification of a DNA fragment, which serves as a mega-primer for the linear amplification of the vector and insert. Here we present several novel applications of the Restriction Free (RF) cloning platform for DNA cloning and mutagenesis. The new applications include simultaneous cloning of several DNA fragments into distinct positions within an expression vector, simultaneous multi-component assembly, and parallel cloning of the same PCR product into a series of different vectors. In addition, we have expanded the application of the RF cloning platform for multiple alterations of the target DNA, including simultaneous multiple-site mutagenesis and simultaneous introduction of deletions and insertions at different positions. We further demonstrate the robustness of the new applications for facilitating recombinant protein expression in the Escherichia coli system. PMID:20600952

  2. Multifunctional proteins revealed by overlapping clustering in protein interaction network

    PubMed Central

    Chapple, Charles E.; Guénoche, Alain; Brun, Christine

    2012-01-01

    Motivation: Multifunctional proteins perform several functions. They are expected to interact specifically with distinct sets of partners, simultaneously or not, depending on the function performed. Current graph clustering methods usually allow a protein to belong to only one cluster, therefore impeding a realistic assignment of multifunctional proteins to clusters. Results: Here, we present Overlapping Cluster Generator (OCG), a novel clustering method which decomposes a network into overlapping clusters and which is, therefore, capable of correct assignment of multifunctional proteins. The principle of OCG is to cover the graph with initial overlapping classes that are iteratively fused into a hierarchy according to an extension of Newman's modularity function. By applying OCG to a human protein–protein interaction network, we show that multifunctional proteins are revealed at the intersection of clusters and demonstrate that the method outperforms other existing methods on simulated graphs and PPI networks. Availability: This software can be downloaded from http://tagc.univ-mrs.fr/welcome/spip.php?rubrique197 Contact: brun@tagc.univ-mrs.fr Supplementary information: Supplementary data are available at Bioinformatics online. PMID:22080466

  3. Dynamic network analysis of protein interactions

    NASA Astrophysics Data System (ADS)

    Almaas, Eivind; Deri, Joya

    2007-03-01

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

  4. Intracellular protein interaction mapping with FRET hybrids

    PubMed Central

    You, Xia; Nguyen, Annalee W.; Jabaiah, Abeer; Sheff, Mark A.; Thorn, Kurt S.; Daugherty, Patrick S.

    2006-01-01

    A quantitative methodology was developed to identify protein interactions in a broad range of cell types by using FRET between fluorescent proteins. Genetic fusions of a target receptor to a FRET acceptor and a large library of candidate peptide ligands to a FRET donor enabled high-throughput optical screening for optimal interaction partners in the cytoplasm of Escherichia coli. Flow cytometric screening identified a panel of peptide ligands capable of recognizing the target receptors in the intracellular environment. For both SH3 and PDZ domain-type target receptors, physiologically meaningful consensus sequences were apparent among the isolated ligands. The relative dissociation constants of interacting partners could be measured directly by using a dilution series of cell lysates containing FRET hybrids, providing a previously undescribed high-throughput approach to rank the affinity of many interaction partners. FRET hybrid interaction screening provides a powerful tool to discover protein ligands in the cellular context with potential applications to a wide variety of eukaryotic cell types. PMID:17130455

  5. Identification of Protein-Protein Interactions and Topologies in Living Cells with Chemical Cross-linking and Mass Spectrometry*S⃞

    PubMed Central

    Zhang, Haizhen; Tang, Xiaoting; Munske, Gerhard R.; Tolic, Nikola; Anderson, Gordon A.; Bruce, James E.

    2009-01-01

    We present results from a novel strategy that enables concurrent identification of protein-protein interactions and topologies in living cells without specific antibodies or genetic manipulations for immuno-/affinity purifications. The strategy consists of (i) a chemical cross-linking reaction: intact cell labeling with a novel class of chemical cross-linkers, protein interaction reporters (PIRs); (ii) two-stage mass spectrometric analysis: stage 1 identification of PIR-labeled proteins and construction of a restricted database by two-dimensional LC/MSMS and stage 2 analysis of PIR-labeled peptides by multiplexed LC/FTICR-MS; and (iii) data analysis: identification of cross-linked peptides and proteins of origin using accurate mass and other constraints. The primary advantage of the PIR approach and distinction from current technology is that protein interactions together with topologies are detected in native biological systems by stabilizing protein complexes with new covalent bonds while the proteins are present in the original cellular environment. Thus, weak or transient interactions or interactions that require properly folded, localized, or membrane-bound proteins can be labeled and identified through the PIR approach. This strategy was applied to Shewanella oneidensis bacterial cells, and initial studies resulted in identification of a set of protein-protein interactions and their contact/binding regions. Furthermore most identified interactions involved membrane proteins, suggesting that the PIR approach is particularly suited for studies of membrane protein-protein interactions, an area under-represented with current widely used approaches. PMID:18936057

  6. Functional module identification in protein interaction networks by interaction patterns

    PubMed Central

    Wang, Yijie; Qian, Xiaoning

    2014-01-01

    Motivation: Identifying functional modules in protein–protein interaction (PPI) networks may shed light on cellular functional organization and thereafter underlying cellular mechanisms. Many existing module identification algorithms aim to detect densely connected groups of proteins as potential modules. However, based on this simple topological criterion of ‘higher than expected connectivity’, those algorithms may miss biologically meaningful modules of functional significance, in which proteins have similar interaction patterns to other proteins in networks but may not be densely connected to each other. A few blockmodel module identification algorithms have been proposed to address the problem but the lack of global optimum guarantee and the prohibitive computational complexity have been the bottleneck of their applications in real-world large-scale PPI networks. Results: In this article, we propose a novel optimization formulation LCP2 (low two-hop conductance sets) using the concept of Markov random walk on graphs, which enables simultaneous identification of both dense and sparse modules based on protein interaction patterns in given networks through searching for LCP2 by random walk. A spectral approximate algorithm SLCP2 is derived to identify non-overlapping functional modules. Based on a bottom-up greedy strategy, we further extend LCP2 to a new algorithm (greedy algorithm for LCP2) GLCP2 to identify overlapping functional modules. We compare SLCP2 and GLCP2 with a range of state-of-the-art algorithms on synthetic networks and real-world PPI networks. The performance evaluation based on several criteria with respect to protein complex prediction, high level Gene Ontology term prediction and especially sparse module detection, has demonstrated that our algorithms based on searching for LCP2 outperform all other compared algorithms. Availability and implementation: All data and code are available at http://www.cse.usf.edu/∼xqian/fmi/slcp2hop

  7. The centrality of cancer proteins in human protein-protein interaction network: a revisit.

    PubMed

    Xiong, Wei; Xie, Luyu; Zhou, Shuigeng; Liu, Hui; Guan, Jihong

    2014-01-01

    Topological analysis of protein-protein interaction (PPI) networks has been widely applied to the investigation on cancer mechanisms. However, there is still a debate on whether cancer proteins exhibit more topological centrality compared to the other proteins in the human PPI network. To resolve this debate, we first identified four sets of human proteins, and then mapped these proteins into the yeast PPI network by homologous genes. Finally, we compared these proteins' properties in human and yeast PPI networks. Experiments over two real datasets demonstrated that cancer proteins tend to have higher degree and smaller clustering coefficient than non-cancer proteins. Experimental results also validated that cancer proteins have larger betweenness centrality compared to the other proteins on the STRING dataset. However, on the BioGRID dataset, the average betweenness centrality of cancer proteins is larger than that of disease and control proteins, but smaller than that of essential proteins. PMID:24878726

  8. Tools for controlling protein interactions with light

    PubMed Central

    Tucker, Chandra L.; Vrana, Justin D.; Kennedy, Matthew J.

    2014-01-01

    Genetically-encoded actuators that allow control of protein-protein interactions with light, termed ‘optical dimerizers’, are emerging as new tools for experimental biology. In recent years, numerous new and versatile dimerizer systems have been developed. Here we discuss the design of optical dimerizer experiments, including choice of a dimerizer system, photoexcitation sources, and coordinate use of imaging reporters. We provide detailed protocols for experiments using two dimerization systems we previously developed, CRY2/CIB and UVR8/UVR8, for use controlling transcription, protein localization, and protein secretion with light. Additionally, we provide instructions and software for constructing a pulse-controlled LED light device for use in experiments requiring extended light treatments. PMID:25181301

  9. Protein Phosphatase 1α Interacting Proteins in the Human Brain

    PubMed Central

    Esteves, Sara L.C.; Domingues, Sara C.; da Cruz e Silva, Odete A.B.; da Cruz e Silva, Edgar F.

    2012-01-01

    Abstract Protein Phosphatase 1 (PP1) is a major serine/threonine-phosphatase whose activity is dependent on its binding to regulatory subunits known as PP1 interacting proteins (PIPs), responsible for targeting PP1 to a specific cellular location, specifying its substrate or regulating its action. Today, more than 200 PIPs have been described involving PP1 in panoply of cellular mechanisms. Moreover, several PIPs have been identified that are tissue and event specific. In addition, the diversity of PP1/PIP complexes can further be achieved by the existence of several PP1 isoforms that can bind preferentially to a certain PIP. Thus, PP1/PIP complexes are highly specific for a particular function in the cell, and as such, they are excellent pharmacological targets. Hence, an in-depth survey was taken to identify specific PP1α PIPs in human brain by a high-throughput Yeast Two-Hybrid approach. Sixty-six proteins were recognized to bind PP1α, 39 being novel PIPs. A large protein interaction databases search was also performed to integrate with the results of the PP1α Human Brain Yeast Two-Hybrid and a total of 246 interactions were retrieved. PMID:22321011

  10. Predicting Disease-Related Proteins Based on Clique Backbone in Protein-Protein Interaction Network

    PubMed Central

    Yang, Lei; Zhao, Xudong; Tang, Xianglong

    2014-01-01

    Network biology integrates different kinds of data, including physical or functional networks and disease gene sets, to interpret human disease. A clique (maximal complete subgraph) in a protein-protein interaction network is a topological module and possesses inherently biological significance. A disease-related clique possibly associates with complex diseases. Fully identifying disease components in a clique is conductive to uncovering disease mechanisms. This paper proposes an approach of predicting disease proteins based on cliques in a protein-protein interaction network. To tolerate false positive and negative interactions in protein networks, extending cliques and scoring predicted disease proteins with gene ontology terms are introduced to the clique-based method. Precisions of predicted disease proteins are verified by disease phenotypes and steadily keep to more than 95%. The predicted disease proteins associated with cliques can partly complement mapping between genotype and phenotype, and provide clues for understanding the pathogenesis of serious diseases. PMID:25013377

  11. The interactions of peripheral membrane proteins with biological membranes

    SciTech Connect

    Johs, Alexander; Whited, A. M.

    2015-01-01

    The interactions of peripheral proteins with membrane surfaces are critical to many biological processes, including signaling, recognition, membrane trafficking, cell division and cell structure. On a molecular level, peripheral membrane proteins can modulate lipid composition, membrane dynamics and protein-protein interactions. Biochemical and biophysical studies have shown that these interactions are in fact highly complex, dominated by several different types of interactions, and have an interdependent effect on both the protein and membrane. Here we examine three major mechanisms underlying the interactions between peripheral membrane proteins and membranes: electrostatic interactions, hydrophobic interactions, and fatty acid modification of proteins. While experimental approaches continue to provide critical insights into specific interaction mechanisms, emerging bioinformatics resources and tools contribute to a systems-level picture of protein-lipid interactions. Through these recent advances, we begin to understand the pivotal role of protein-lipid interactions underlying complex biological functions at membrane interfaces.

  12. The interactions of peripheral membrane proteins with biological membranes

    DOE PAGESBeta

    Johs, Alexander; Whited, A. M.

    2015-01-01

    The interactions of peripheral proteins with membrane surfaces are critical to many biological processes, including signaling, recognition, membrane trafficking, cell division and cell structure. On a molecular level, peripheral membrane proteins can modulate lipid composition, membrane dynamics and protein-protein interactions. Biochemical and biophysical studies have shown that these interactions are in fact highly complex, dominated by several different types of interactions, and have an interdependent effect on both the protein and membrane. Here we examine three major mechanisms underlying the interactions between peripheral membrane proteins and membranes: electrostatic interactions, hydrophobic interactions, and fatty acid modification of proteins. While experimental approachesmore » continue to provide critical insights into specific interaction mechanisms, emerging bioinformatics resources and tools contribute to a systems-level picture of protein-lipid interactions. Through these recent advances, we begin to understand the pivotal role of protein-lipid interactions underlying complex biological functions at membrane interfaces.« less

  13. Using support vector machine for improving protein-protein interaction prediction utilizing domain interactions

    SciTech Connect

    Singhal, Mudita; Shah, Anuj R.; Brown, Roslyn N.; Adkins, Joshua N.

    2010-10-02

    Understanding protein interactions is essential to gain insights into the biological processes at the whole cell level. The high-throughput experimental techniques for determining protein-protein interactions (PPI) are error prone and expensive with low overlap amongst them. Although several computational methods have been proposed for predicting protein interactions there is definite room for improvement. Here we present DomainSVM, a predictive method for PPI that uses computationally inferred domain-domain interaction values in a Support Vector Machine framework to predict protein interactions. DomainSVM method utilizes evidence of multiple interacting domains to predict a protein interaction. It outperforms existing methods of PPI prediction by achieving very high explanation ratios, precision, specificity, sensitivity and F-measure values in a 10 fold cross-validation study conducted on the positive and negative PPIs in yeast. A Functional comparison study using GO annotations on the positive and the negative test sets is presented in addition to discussing novel PPI predictions in Salmonella Typhimurium.

  14. Phenylboronate chromatography selectively separates glycoproteins through the manipulation of electrostatic, charge transfer, and cis-diol interactions.

    PubMed

    Carvalho, Rimenys J; Woo, James; Aires-Barros, M Raquel; Cramer, Steven M; Azevedo, Ana M

    2014-10-01

    Phenylboronate chromatography (PBC) has been applied for several years, however details regarding the mechanisms of interactions between the ligand and biomolecules are still scarce. The goal of this work is to investigate the various chemical interactions between proteins and their ligands, using a protein library containing both glycosylated and nonglycosylated proteins. Differences in the adsorption of these proteins over a pH range from 4 to 9 were related to two main properties: charge and presence of glycans. Acidic or neutral proteins were strongly adsorbed below pH 8 although the uncharged trigonal form of phenylboronate (PB) is less susceptible to forming electrostatic and cis-diol interactions with proteins. The glycosylated proteins were only adsorbed above pH 8 when the electrostatic repulsion between the boronate anion and the protein surface was mitigated (at 200 mM NaCl). All basic proteins were highly adsorbed above pH 8 with PB also acting as a cation-exchanger with binding occurring through electrostatic interactions. Batch adsorption performed at acidic conditions in the presence of Lewis base showed that charge-transfer interactions are critical for protein retention. This study demonstrates the multimodal interaction of PBC, which can be a selective tool for separation of different classes of proteins. PMID:25130283

  15. Manipulation of cellular light from green fluorescent protein by a femtosecond laser

    NASA Astrophysics Data System (ADS)

    He, Hao; Li, Shiyang; Wang, Shaoyang; Hu, Minglie; Cao, Youjia; Wang, Chingyue

    2012-10-01

    Green fluorescent protein (GFP) is one of the most widely studied and exploited proteins in biochemistry and cell biology. It emits fluorescence following optical excitation, which is usually provided by a laser. Here, we report that fluorescence from enhanced GFP can be `turned off' by exposing cells to laser light. A short flash of femtosecond laser light is shown to deplete calcium in the endoplasmic reticulum of cells. Calcium-release-activated calcium channels are then activated by stromal interaction molecule 1 (STIM1). The rise in intracellular Ca2+ depolarizes mitochondria and increases the leakage of reactive oxygen species, which then permanently bleach the GFP. This controllable optical scheme for reactive oxygen species generation can also be used to modulate the photoconversion of GFP fluorescence from green to red emission and provide a mechanism for influencing cellular molecular dynamics.

  16. Ultra-fast optical manipulation of single proteins binding to the actin cytoskeleton

    NASA Astrophysics Data System (ADS)

    Capitanio, Marco; Gardini, Lucia; Pavone, Francesco Saverio

    2014-02-01

    In the last decade, forces and mechanical stresses acting on biological systems are emerging as regulatory factors essential for cell life. Emerging evidences indicate that factors such as applied forces or the rigidity of the extracellular matrix (ECM) determine the shape and function of cells and organisms1. Classically, the regulation of biological systems is described through a series of biochemical signals and enzymatic reactions, which direct the processes and cell fate. However, mechanotransduction, i.e. the conversion of mechanical forces into biochemical and biomolecular signals, is at the basis of many biological processes fundamental for the development and differentiation of cells, for their correct function and for the development of pathologies. We recently developed an in vitro system that allows the investigation of force-dependence of the interaction of proteins binding the actin cytoskeleton, at the single molecule level. Our system displays a delay of only ~10 μs between formation of the molecular bond and application of the force and is capable of detecting interactions as short as 100 μs. Our assay allows direct measurements of load-dependence of lifetimes of single molecular bonds and conformational changes of single proteins and molecular motors. We demonstrate our technique on molecular motors, using myosin II from fast skeletal muscle and on protein-DNA interaction, specifically on Lactose repressor (LacI). The apparatus is stabilized to less than 1 nm with both passive and active stabilization, allowing resolving specific binding regions along the actin filament and DNA molecule. Our technique extends single-molecule force-clamp spectroscopy to molecular complexes that have been inaccessible up to now, opening new perspectives for the investigation of the effects of forces on biological processes.

  17. Ribo-Proteomics Approach to Profile RNA-Protein and Protein-Protein Interaction Networks.

    PubMed

    Yeh, Hsin-Sung; Chang, Jae-Woong; Yong, Jeongsik

    2016-01-01

    Characterizing protein-protein and protein-RNA interaction networks is a fundamental step to understanding the function of an RNA-binding protein. In many cases, these interactions are transient and highly dynamic. Therefore, capturing stable as well as transient interactions in living cells for the identification of protein-binding partners and the mapping of RNA-binding sequences is key to a successful establishment of the molecular interaction network. In this chapter, we will describe a method for capturing the molecular interactions in living cells using formaldehyde as a crosslinker and enriching a specific RNA-protein complex from cell extracts followed by mass spectrometry and Next-Gen sequencing analyses. PMID:26965265

  18. Michigan molecular interactions r2: from interacting proteins to pathways.

    PubMed

    Tarcea, V Glenn; Weymouth, Terry; Ade, Alex; Bookvich, Aaron; Gao, Jing; Mahavisno, Vasudeva; Wright, Zach; Chapman, Adriane; Jayapandian, Magesh; Ozgür, Arzucan; Tian, Yuanyuan; Cavalcoli, Jim; Mirel, Barbara; Patel, Jignesh; Radev, Dragomir; Athey, Brian; States, David; Jagadish, H V

    2009-01-01

    Molecular interaction data exists in a number of repositories, each with its own data format, molecule identifier and information coverage. Michigan molecular interactions (MiMI) assists scientists searching through this profusion of molecular interaction data. The original release of MiMI gathered data from well-known protein interaction databases, and deep merged this information while keeping track of provenance. Based on the feedback received from users, MiMI has been completely redesigned. This article describes the resulting MiMI Release 2 (MiMIr2). New functionality includes extension from proteins to genes and to pathways; identification of highlighted sentences in source publications; seamless two-way linkage with Cytoscape; query facilities based on MeSH/GO terms and other concepts; approximate graph matching to find relevant pathways; support for querying in bulk; and a user focus-group driven interface design. MiMI is part of the NIH's; National Center for Integrative Biomedical Informatics (NCIBI) and is publicly available at: http://mimi.ncibi.org. PMID:18978014

  19. Protein-protein interactions of mitochondrial-associated protein via bioluminescence resonance energy transfer

    PubMed Central

    Koshiba, Takumi

    2015-01-01

    Protein-protein interactions are essential biological reactions occurring at inter- and intra-cellular levels. The analysis of their mechanism is generally required in order link to understand their various cellular functions. Bioluminescence resonance energy transfer (BRET), which is based on an enzymatic activity of luciferase, is a useful tool for investigating protein-protein interactions in live cells. The combination of the BRET system and biomolecular fluorescence complementation (BiFC) would provide us a better understanding of the hetero-oligomeric structural states of protein complexes. In this review, we discuss the application of BRET to the protein-protein interactions of mitochondrial-associated proteins and discuss its physiological relevance. PMID:27493852

  20. Turning the spotlight on protein-lipid interactions in cells

    PubMed Central

    Peng, Tao; Yuan, Xiaoqiu; Hang, Howard C.

    2014-01-01

    Protein function is largely dependent on coordinated and dynamic interactions of the protein with biomolecules including other proteins, nucleic acids and lipids. While powerful methods for global profiling of protein-protein and protein-nucleic acid interactions are available, proteome-wide mapping of protein-lipid interactions is still challenging and rarely performed. The emergence of bifunctional lipid probes with photoactivatable and clickable groups offers new chemical tools for globally profiling protein-lipid interactions under cellular contexts. In this review, we summarize recent advances in the development of bifunctional lipid probes for studying protein-lipid interactions. We also highlight how in vivo photocrosslinking reactions contribute to the characterization of lipid-binding proteins and lipidation-mediated protein-protein interactions. PMID:25129056

  1. Schizophrenia interactome with 504 novel protein-protein interactions.

    PubMed

    Ganapathiraju, Madhavi K; Thahir, Mohamed; Handen, Adam; Sarkar, Saumendra N; Sweet, Robert A; Nimgaonkar, Vishwajit L; Loscher, Christine E; Bauer, Eileen M; Chaparala, Srilakshmi

    2016-01-01

    Genome-wide association studies of schizophrenia (GWAS) have revealed the role of rare and common genetic variants, but the functional effects of the risk variants remain to be understood. Protein interactome-based studies can facilitate the study of molecular mechanisms by which the risk genes relate to schizophrenia (SZ) genesis, but protein-protein interactions (PPIs) are unknown for many of the liability genes. We developed a computational model to discover PPIs, which is found to be highly accurate according to computational evaluations and experimental validations of selected PPIs. We present here, 365 novel PPIs of liability genes identified by the SZ Working Group of the Psychiatric Genomics Consortium (PGC). Seventeen genes that had no previously known interactions have 57 novel interactions by our method. Among the new interactors are 19 drug targets that are targeted by 130 drugs. In addition, we computed 147 novel PPIs of 25 candidate genes investigated in the pre-GWAS era. While there is little overlap between the GWAS genes and the pre-GWAS genes, the interactomes reveal that they largely belong to the same pathways, thus reconciling the apparent disparities between the GWAS and prior gene association studies. The interactome including 504 novel PPIs overall, could motivate other systems biology studies and trials with repurposed drugs. The PPIs are made available on a webserver, called Schizo-Pi at http://severus.dbmi.pitt.edu/schizo-pi with advanced search capabilities. PMID:27336055

  2. Mapping Protein Interactions between Dengue Virus and Its Human and Insect Hosts

    PubMed Central

    Doolittle, Janet M.; Gomez, Shawn M.

    2011-01-01

    Background Dengue fever is an increasingly significant arthropod-borne viral disease, with at least 50 million cases per year worldwide. As with other viral pathogens, dengue virus is dependent on its host to perform the bulk of functions necessary for viral survival and replication. To be successful, dengue must manipulate host cell biological processes towards its own ends, while avoiding elimination by the immune system. Protein-protein interactions between the virus and its host are one avenue through which dengue can connect and exploit these host cellular pathways and processes. Methodology/Principal Findings We implemented a computational approach to predict interactions between Dengue virus (DENV) and both of its hosts, Homo sapiens and the insect vector Aedes aegypti. Our approach is based on structural similarity between DENV and host proteins and incorporates knowledge from the literature to further support a subset of the predictions. We predict over 4,000 interactions between DENV and humans, as well as 176 interactions between DENV and A. aegypti. Additional filtering based on shared Gene Ontology cellular component annotation reduced the number of predictions to approximately 2,000 for humans and 18 for A. aegypti. Of 19 experimentally validated interactions between DENV and humans extracted from the literature, this method was able to predict nearly half (9). Additional predictions suggest specific interactions between virus and host proteins relevant to interferon signaling, transcriptional regulation, stress, and the unfolded protein response. Conclusions/Significance Dengue virus manipulates cellular processes to its advantage through specific interactions with the host's protein interaction network. The interaction networks presented here provide a set of hypothesis for further experimental investigation into the DENV life cycle as well as potential therapeutic targets. PMID:21358811

  3. Notable Aspects of Glycan-Protein Interactions

    PubMed Central

    Cohen, Miriam

    2015-01-01

    This mini review highlights several interesting aspects of glycan-mediated interactions that are common between cells, bacteria, and viruses. Glycans are ubiquitously found on all living cells, and in the extracellular milieu of multicellular organisms. They are known to mediate initial binding and recognition events of both immune cells and pathogens with their target cells or tissues. The host target tissues are hidden under a layer of secreted glycosylated decoy targets. In addition, pathogens can utilize and display host glycans to prevent identification as foreign by the host’s immune system (molecular mimicry). Both the host and pathogens continually evolve. The host evolves to prevent infection and the pathogens evolve to evade host defenses. Many pathogens express both glycan-binding proteins and glycosidases. Interestingly, these proteins are often located at the tip of elongated protrusions in bacteria, or in the leading edge of the cell. Glycan-protein interactions have low affinity and, as a result, multivalent interactions are often required to achieve biologically relevant binding. These enable dynamic forms of adhesion mechanisms, reviewed here, and include rolling (cells), stick and roll (bacteria) or surfacing (viruses). PMID:26340640

  4. Notable Aspects of Glycan-Protein Interactions.

    PubMed

    Cohen, Miriam

    2015-01-01

    This mini review highlights several interesting aspects of glycan-mediated interactions that are common between cells, bacteria, and viruses. Glycans are ubiquitously found on all living cells, and in the extracellular milieu of multicellular organisms. They are known to mediate initial binding and recognition events of both immune cells and pathogens with their target cells or tissues. The host target tissues are hidden under a layer of secreted glycosylated decoy targets. In addition, pathogens can utilize and display host glycans to prevent identification as foreign by the host's immune system (molecular mimicry). Both the host and pathogens continually evolve. The host evolves to prevent infection and the pathogens evolve to evade host defenses. Many pathogens express both glycan-binding proteins and glycosidases. Interestingly, these proteins are often located at the tip of elongated protrusions in bacteria, or in the leading edge of the cell. Glycan-protein interactions have low affinity and, as a result, multivalent interactions are often required to achieve biologically relevant binding. These enable dynamic forms of adhesion mechanisms, reviewed here, and include rolling (cells), stick and roll (bacteria) or surfacing (viruses). PMID:26340640

  5. Identification of essential proteins based on ranking edge-weights in protein-protein interaction networks.

    PubMed

    Wang, Yan; Sun, Huiyan; Du, Wei; Blanzieri, Enrico; Viero, Gabriella; Xu, Ying; Liang, Yanchun

    2014-01-01

    Essential proteins are those that are indispensable to cellular survival and development. Existing methods for essential protein identification generally rely on knock-out experiments and/or the relative density of their interactions (edges) with other proteins in a Protein-Protein Interaction (PPI) network. Here, we present a computational method, called EW, to first rank protein-protein interactions in terms of their Edge Weights, and then identify sub-PPI-networks consisting of only the highly-ranked edges and predict their proteins as essential proteins. We have applied this method to publicly-available PPI data on Saccharomyces cerevisiae (Yeast) and Escherichia coli (E. coli) for essential protein identification, and demonstrated that EW achieves better performance than the state-of-the-art methods in terms of the precision-recall and Jackknife measures. The highly-ranked protein-protein interactions by our prediction tend to be biologically significant in both the Yeast and E. coli PPI networks. Further analyses on systematically perturbed Yeast and E. coli PPI networks through randomly deleting edges demonstrate that the proposed method is robust and the top-ranked edges tend to be more associated with known essential proteins than the lowly-ranked edges. PMID:25268881

  6. Heparan sulfate and heparin interactions with proteins

    PubMed Central

    Meneghetti, Maria C. Z.; Hughes, Ashley J.; Rudd, Timothy R.; Nader, Helena B.; Powell, Andrew K.; Yates, Edwin A.; Lima, Marcelo A.

    2015-01-01

    Heparan sulfate (HS) polysaccharides are ubiquitous components of the cell surface and extracellular matrix of all multicellular animals, whereas heparin is present within mast cells and can be viewed as a more sulfated, tissue-specific, HS variant. HS and heparin regulate biological processes through interactions with a large repertoire of proteins. Owing to these interactions and diverse effects observed during in vitro, ex vivo and in vivo experiments, manifold biological/pharmacological activities have been attributed to them. The properties that have been thought to bestow protein binding and biological activity upon HS and heparin vary from high levels of sequence specificity to a dependence on charge. In contrast to these opposing opinions, we will argue that the evidence supports both a level of redundancy and a degree of selectivity in the structure–activity relationship. The relationship between this apparent redundancy, the multi-dentate nature of heparin and HS polysaccharide chains, their involvement in protein networks and the multiple binding sites on proteins, each possessing different properties, will also be considered. Finally, the role of cations in modulating HS/heparin activity will be reviewed and some of the implications for structure–activity relationships and regulation will be discussed. PMID:26289657

  7. The influence of protein-protein interactions on the organization of proteins within thylakoid membranes.

    PubMed

    Tremmel, I G; Weis, E; Farquhar, G D

    2005-04-01

    The influence of attractive protein-protein interactions on the organization of photosynthetic proteins within the thylakoid membrane was investigated. Protein-protein interactions were simulated using Monte Carlo techniques and the influence of different interaction energies was examined. It was found that weak interactions led to protein clusters whereas strong interactions led to ramified chains. An optimum curve for the relationship between interaction energy and the number of contact sites emerged. With increasing particle densities the effect decreased. In a mixture of interacting and noninteracting particles the distance between the noninteracting particles was increased and there seemed to be much more free space around them. In thylakoids, this could lead to a more homogeneous distribution of the noninteracting but rate-limiting cytochrome bf complexes. Due to the increased free space between cytochrome bf, obstruction of binding sites--occurring unavoidably in a random distribution--may be drastically reduced. Furthermore, protein-protein interactions in thylakoids may lead to a decrease in plastoquinone diffusion. PMID:15665125

  8. Imaging Protein Protein Interactions inside Living Cells via Interaction-Dependent Fluorophore Ligation

    PubMed Central

    Slavoff, Sarah A.; Liu, Daniel S.; Cohen, Justin D.; Ting, Alice Y.

    2012-01-01

    We report a new method, Interaction-Dependent PRobe Incorporation Mediated by Enzymes, or ID-PRIME, for imaging protein protein interactions (PPIs) inside living cells. ID-PRIME utilizes a mutant of Escherichia coli lipoic acid ligase, LplAW37V, which can catalyze the covalent ligation of a coumarin fluorophore onto a peptide recognition sequence called LAP1. The affinity between the ligase and LAP1 is tuned such that, when each is fused to a protein partner of interest, LplAW37V labels LAP1 with coumarin only when the protein partners to which they are fused bring them together. Coumarin labeling in the absence of such interaction is low or undetectable. Characterization of ID-PRIME in living mammalian cells shows that multiple protein protein interactions can be imaged (FRB FKBP, Fos Jun, and neuroligin PSD-95), with as little as 10 min of coumarin treatment. The signal intensity and detection sensitivity are similar to those of the widely used fluorescent protein complementation technique (BiFC) for PPI detection, without the disadvantage of irreversible complex trapping. ID-PRIME provides a powerful and complementary approach to existing methods for visualization of PPIs in living cells with spatial and temporal resolution. PMID:22098454

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

    PubMed Central

    2014-01-01

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

  10. 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

  11. An Interactive, Versatile, Three-Dimensional Display, Manipulation and Plotting System for Biomedical Research

    ERIC Educational Resources Information Center

    Feldmann, Richard J.; And Others

    1972-01-01

    Computer graphics provides a valuable tool for the representation and a better understanding of structures, both small and large. Accurate and rapid construction, manipulation, and plotting of structures, such as macromolecules as complex as hemoglobin, are performed by a collection of computer programs and a time-sharing computer. (21 references)…

  12. Light-Scattering Studies of Protein Solutions: Role of Hydration in Weak Protein-Protein Interactions

    PubMed Central

    Paliwal, A.; Asthagiri, D.; Abras, D.; Lenhoff, A. M.; Paulaitis, M. E.

    2005-01-01

    We model the hydration contribution to short-range electrostatic/dispersion protein interactions embodied in the osmotic second virial coefficient, B2, by adopting a quasi-chemical description in which water molecules associated with the protein are identified through explicit molecular dynamics simulations. These water molecules reduce the surface complementarity of highly favorable short-range interactions, and therefore can play an important role in mediating protein-protein interactions. Here we examine this quasi-chemical view of hydration by predicting the interaction part of B2 and comparing our results with those derived from light-scattering measurements of B2 for staphylococcal nuclease, lysozyme, and chymotrypsinogen at 25°C as a function of solution pH and ionic strength. We find that short-range protein interactions are influenced by water molecules strongly associated with a relatively small fraction of the protein surface. However, the effect of these strongly associated water molecules on the surface complementarity of short-range protein interactions is significant, and must be taken into account for an accurate description of B2. We also observe remarkably similar hydration behavior for these proteins despite substantial differences in their three-dimensional structures and spatial charge distributions, suggesting a general characterization of protein hydration. PMID:15980182

  13. Algorithmic approaches to protein-protein interaction site prediction.

    PubMed

    Aumentado-Armstrong, Tristan T; Istrate, Bogdan; Murgita, Robert A

    2015-01-01

    Interaction sites on protein surfaces mediate virtually all biological activities, and their identification holds promise for disease treatment and drug design. Novel algorithmic approaches for the prediction of these sites have been produced at a rapid rate, and the field has seen significant advancement over the past decade. However, the most current methods have not yet been reviewed in a systematic and comprehensive fashion. Herein, we describe the intricacies of the biological theory, datasets, and features required for modern protein-protein interaction site (PPIS) prediction, and present an integrative analysis of the state-of-the-art algorithms and their performance. First, the major sources of data used by predictors are reviewed, including training sets, evaluation sets, and methods for their procurement. Then, the features employed and their importance in the biological characterization of PPISs are explored. This is followed by a discussion of the methodologies adopted in contemporary prediction programs, as well as their relative performance on the datasets most recently used for evaluation. In addition, the potential utility that PPIS identification holds for rational drug design, hotspot prediction, and computational molecular docking is described. Finally, an analysis of the most promising areas for future development of the field is presented. PMID:25713596

  14. Deciphering Supramolecular Structures with Protein-Protein Interaction Network Modeling

    PubMed Central

    Tsuji, Toshiyuki; Yoda, Takao; Shirai, Tsuyoshi

    2015-01-01

    Many biological molecules are assembled into supramolecules that are essential to perform complicated functions in the cell. However, experimental information about the structures of supramolecules is not sufficient at this point. We developed a method of predicting and modeling the structures of supramolecules in a biological network by combining structural data of the Protein Data Bank (PDB) and interaction data in IntAct databases. Templates for binary complexes in IntAct were extracted from PDB. Modeling was attempted by assembling binary complexes with superposed shared subunits. A total of 3,197 models were constructed, and 1,306 (41% of the total) contained at least one subunit absent from experimental structures. The models also suggested 970 (25% of the total) experimentally undetected subunit interfaces, and 41 human disease-related amino acid variants were mapped onto these model-suggested interfaces. The models demonstrated that protein-protein interaction network modeling is useful to fill the information gap between biological networks and structures. PMID:26549015

  15. Targeting protein-protein interactions as an anticancer strategy

    PubMed Central

    Ivanov, Andrei A.; Khuri, Fadlo R.; Fu, Haian

    2013-01-01

    The emergence and convergence of cancer genomics, targeted therapies, and network oncology have significantly expanded the landscape of protein-protein interaction (PPI) networks in cancer for therapeutic discovery. Extensive biological and clinical investigations have led to the identification of protein interaction hubs and nodes that are critical for the acquisition and maintaining characteristics of cancer essential for cell transformation. Such cancer enabling PPIs have become promising therapeutic targets. With technological advances in PPI modulator discovery and validation of PPI-targeting agents in clinical settings, targeting PPI interfaces as an anticancer strategy has become a reality. Future research directed at genomics-based PPI target discovery, PPI interface characterization, PPI-focused chemical library design, and patient-genomic subpopulation-driven clinical studies is expected to accelerate the development of the next generation of PPI-based anticancer agents for personalized precision medicine. Here we briefly review prominent PPIs that mediate cancer-acquired properties, highlight recognized challenges and promising clinical results in targeting PPIs, and outline emerging opportunities. PMID:23725674

  16. Parallel Force Assay for Protein-Protein Interactions

    PubMed Central

    Aschenbrenner, Daniela; Pippig, Diana A.; Klamecka, Kamila; Limmer, Katja; Leonhardt, Heinrich; Gaub, Hermann E.

    2014-01-01

    Quantitative proteome research is greatly promoted by high-resolution parallel format assays. A characterization of protein complexes based on binding forces offers an unparalleled dynamic range and allows for the effective discrimination of non-specific interactions. Here we present a DNA-based Molecular Force Assay to quantify protein-protein interactions, namely the bond between different variants of GFP and GFP-binding nanobodies. We present different strategies to adjust the maximum sensitivity window of the assay by influencing the binding strength of the DNA reference duplexes. The binding of the nanobody Enhancer to the different GFP constructs is compared at high sensitivity of the assay. Whereas the binding strength to wild type and enhanced GFP are equal within experimental error, stronger binding to superfolder GFP is observed. This difference in binding strength is attributed to alterations in the amino acids that form contacts according to the crystal structure of the initial wild type GFP-Enhancer complex. Moreover, we outline the potential for large-scale parallelization of the assay. PMID:25546146

  17. The critical protein interactions and structures that elicit growth deregulation in cancer and viral replication

    PubMed Central

    Ou, Horng D.; May, Andrew P.

    2010-01-01

    One of the greatest challenges in biomedicine is to define the critical targets and network interactions that are subverted to elicit growth deregulation in human cells. Understanding and developing rational treatments for cancer requires a definition of the key molecular targets and how they interact to elicit the complex growth deregulation phenotype. Viral proteins provide discerning and powerful probes to understand both how cells work and how they can be manipulated using a minimal number of components. The small DNA viruses have evolved to target inherent weaknesses in cellular protein interaction networks to hijack the cellular DNA and protein replication machinery. In the battle to escape the inevitability of senescence and programmed cell death, cancers have converged on similar mechanisms, through the acquisition and selection of somatic mutations that drive unchecked cellular replication in tumors. Understanding the dynamic mechanisms through which a minimal number of viral proteins promote host cells to undergo unscheduled and pathological replication is a powerful strategy to identify critical targets that are also disrupted in cancer. Viruses can therefore be used as tools to probe the system-wide protein-protein interactions and structures that drive growth deregulation in human cells. Ultimately this can provide a path for developing system context-dependent therapeutics. This review will describe ongoing experimental approaches using viruses to study pathways deregulated in cancer, with a particular focus on viral cellular protein-protein interactions and structures. PMID:21061422

  18. The critical protein interactions and structures that elicit growth deregulation in cancer and viral replication.

    PubMed

    Ou, Horng D; May, Andrew P; O'Shea, Clodagh C

    2011-01-01

    One of the greatest challenges in biomedicine is to define the critical targets and network interactions that are subverted to elicit growth deregulation in human cells. Understanding and developing rational treatments for cancer requires a definition of the key molecular targets and how they interact to elicit the complex growth deregulation phenotype. Viral proteins provide discerning and powerful probes to understand both how cells work and how they can be manipulated using a minimal number of components. The small DNA viruses have evolved to target inherent weaknesses in cellular protein interaction networks to hijack the cellular DNA and protein replication machinery. In the battle to escape the inevitability of senescence and programmed cell death, cancers have converged on similar mechanisms, through the acquisition and selection of somatic mutations that drive unchecked cellular replication in tumors. Understanding the dynamic mechanisms through which a minimal number of viral proteins promote host cells to undergo unscheduled and pathological replication is a powerful strategy to identify critical targets that are also disrupted in cancer. Viruses can therefore be used as tools to probe the system-wide protein-protein interactions and structures that drive growth deregulation in human cells. Ultimately this can provide a path for developing system context-dependent therapeutics. This review will describe ongoing experimental approaches using viruses to study pathways deregulated in cancer, with a particular focus on viral cellular protein-protein interactions and structures. PMID:21061422

  19. Module organization and variance in protein-protein interaction networks

    PubMed Central

    Lin, Chun-Yu; Lee, Tsai-Ling; Chiu, Yi-Yuan; Lin, Yi-Wei; Lo, Yu-Shu; Lin, Chih-Ta; Yang, Jinn-Moon

    2015-01-01

    A module is a group of closely related proteins that act in concert to perform specific biological functions through protein–protein interactions (PPIs) that occur in time and space. However, the underlying module organization and variance remain unclear. In this study, we collected module templates to infer respective module families, including 58,041 homologous modules in 1,678 species, and PPI families using searches of complete genomic database. We then derived PPI evolution scores and interface evolution scores to describe the module elements, including core and ring components. Functions of core components were highly correlated with those of essential genes. In comparison with ring components, core proteins/PPIs were conserved across multiple species. Subsequently, protein/module variance of PPI networks confirmed that core components form dynamic network hubs and play key roles in various biological functions. Based on the analyses of gene essentiality, module variance, and gene co-expression, we summarize the observations of module organization and variance as follows: 1) a module consists of core and ring components; 2) core components perform major biological functions and collaborate with ring components to execute certain functions in some cases; 3) core components are more conserved and essential during organizational changes in different biological states or conditions. PMID:25797237

  20. From Topology to Phenotype in Protein-Protein Interaction Networks

    NASA Astrophysics Data System (ADS)

    Pržulj, Nataša

    We have recently witnessed an explosion in biological network data along with the development of computational approaches for their analyses. This new interdisciplinary research area is an integral part of systems biology, promising to provide new insights into organizational principles of life, as well as into evolution and disease. However, there is a danger that the area might become hindered by several emerging issues. In particular, there is typically a weak link between biological and computational scientists, resulting in the use of simple computational techniques of limited potential to explain these complex biological data. Hence, there is a danger that the community might view the topological features of network data as mere statistics, ignoring the value of the information contained in these data. This might result in the imposition of scientific doctrines, such as scale-free-centric (on the modelling side) and genome-centric (on the biological side) opinions onto this nascent research area. In this chapter, we take a network science perspective and present a brief, high-level overview of the area, commenting on possible challenges ahead. We focus on protein-protein interaction networks (PINs) in which nodes correspond to proteins in a cell and edges to physical bindings between the proteins.

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

    PubMed Central

    Samuels, M; Deshpande, G; Schedl, P

    1998-01-01

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

  2. Slim-Filter: an interactive windows-based application for illumina genome analyzer data assessment and manipulation

    PubMed Central

    2012-01-01

    Background The emergence of Next Generation Sequencing technologies has made it possible for individual investigators to generate gigabases of sequencing data per week. Effective analysis and manipulation of these data is limited due to large file sizes, so even simple tasks such as data filtration and quality assessment have to be performed in several steps. This requires (potentially problematic) interaction between the investigator and a bioinformatics/computational service provider. Furthermore, such services are often performed using specialized computational facilities. Results We present a Windows-based application, Slim-Filter designed to interactively examine the statistical properties of sequencing reads produced by Illumina Genome Analyzer and to perform a broad spectrum of data manipulation tasks including: filtration of low quality and low complexity reads; filtration of reads containing undesired subsequences (such as parts of adapters and PCR primers used during the sample and sequencing libraries preparation steps); excluding duplicated reads (while keeping each read’s copy number information in a specialized data format); and sorting reads by copy numbers allowing for easy access and manual editing of the resulting files. Slim-Filter is organized as a sequence of windows summarizing the statistical properties of the reads. Each data manipulation step has roll-back abilities, allowing for return to previous steps of the data analysis process. Slim-Filter is written in C++ and is compatible with fasta, fastq, and specialized AS file formats presented in this manuscript. Setup files and a user’s manual are available for download at the supplementary web site ( https://www.bioinfo.uh.edu/Slim_Filter/). Conclusion The presented Windows-based application has been developed with the goal of providing individual investigators with integrated sequencing reads analysis, curation, and manipulation capabilities. PMID:22800377

  3. Developing algorithms for predicting protein-protein interactions of homology modeled proteins.

    SciTech Connect

    Martin, Shawn Bryan; Sale, Kenneth L.; Faulon, Jean-Loup Michel; Roe, Diana C.

    2006-01-01

    The goal of this project was to examine the protein-protein docking problem, especially as it relates to homology-based structures, identify the key bottlenecks in current software tools, and evaluate and prototype new algorithms that may be developed to improve these bottlenecks. This report describes the current challenges in the protein-protein docking problem: correctly predicting the binding site for the protein-protein interaction and correctly placing the sidechains. Two different and complementary approaches are taken that can help with the protein-protein docking problem. The first approach is to predict interaction sites prior to docking, and uses bioinformatics studies of protein-protein interactions to predict theses interaction site. The second approach is to improve validation of predicted complexes after docking, and uses an improved scoring function for evaluating proposed docked poses, incorporating a solvation term. This scoring function demonstrates significant improvement over current state-of-the art functions. Initial studies on both these approaches are promising, and argue for full development of these algorithms.

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

    PubMed

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

    2016-08-16

    Capillary electrophoresis (CE) has been identified as a useful platform for detecting, quantifying, and screening for modulators of protein-protein interactions (PPIs). In this method, one protein binding partner is labeled with a fluorophore, the protein binding partners are mixed, and then, the complex is separated from free protein to allow direct determination of bound to free ratios. Although it possesses many advantages for PPI studies, the method is limited by the need to have separation conditions that both prevent protein adsorption to capillary and maintain protein interactions during the separation. In this work, we use protein cross-linking capillary electrophoresis (PXCE) to overcome this limitation. In PXCE, the proteins are cross-linked under binding conditions and then separated. This approach eliminates the need to maintain noncovalent interactions during electrophoresis and facilitates method development. We report PXCE methods for an antibody-antigen interaction and heterodimer and homodimer heat shock protein complexes. Complexes are cross-linked by short treatments with formaldehyde after reaching binding equilibrium. Cross-linked complexes are separated by electrophoretic mobility using free solution CE or by size using sieving electrophoresis of SDS complexes. The method gives good quantitative results; e.g., a lysozyme-antibody interaction was found to have Kd = 24 ± 3 nM by PXCE and Kd = 17 ± 2 nM using isothermal calorimetry (ITC). Heat shock protein 70 (Hsp70) in complex with bcl2 associated athanogene 3 (Bag3) was found to have Kd = 25 ± 5 nM by PXCE which agrees with Kd values reported without cross-linking. Hsp70-Bag3 binding site mutants and small molecule inhibitors of Hsp70-Bag3 were characterized by PXCE with good agreement to inhibitory constants and IC50 values obtained by a bead-based flow cytometry protein interaction assay (FCPIA). PXCE allows rapid method development for quantitative analysis of PPIs. PMID:27434096

  5. Ortholog-based protein-protein interaction prediction and its application to inter-species interactions

    PubMed Central

    Lee, Sheng-An; Chan, Cheng-hsiung; Tsai, Chi-Hung; Lai, Jin-Mei; Wang, Feng-Sheng; Kao, Cheng-Yan; Huang, Chi-Ying F

    2008-01-01

    Background The rapid growth of protein-protein interaction (PPI) data has led to the emergence of PPI network analysis. Despite advances in high-throughput techniques, the interactomes of several model organisms are still far from complete. Therefore, it is desirable to expand these interactomes with ortholog-based and other methods. Results Orthologous pairs of 18 eukaryotic species were expanded and merged with experimental PPI datasets. The contributions of interologs from each species were evaluated. The expanded orthologous pairs enable the inference of interologs for various species. For example, more than 32,000 human interactions can be predicted. The same dataset has also been applied to the prediction of host-pathogen interactions. PPIs between P. falciparum calmodulin and several H. sapiens proteins are predicted, and these interactions may contribute to the maintenance of host cell Ca2+ concentration. Using comparisons with Bayesian and structure-based approaches, interactions between putative HSP40 homologs of P. falciparum and the H. sapiens TNF receptor associated factor family are revealed, suggesting a role for these interactions in the interference of the human immune response to P. falciparum. Conclusion The PPI datasets are available from POINT and POINeT . Further development of methods to predict host-pathogen interactions should incorporate multiple approaches in order to improve sensitivity, and should facilitate the identification of targets for drug discovery and design. PMID:19091010

  6. Detection and identification of protein interactions of S100 proteins by ProteinChip technology.

    PubMed

    Lehmann, Roland; Melle, Christian; Escher, Niko; von Eggeling, Ferdinand

    2005-01-01

    The aim of this work was to establish an approach for identification of protein interactions. This assay used an anti-S100A8 antibody coupled on beads and incubated with cell extract. The bead eluates were analyzed using ProteinChip technology and subsequently subjected to an appropriate digestion. Molecular masses of digestion fragments were determined by SELDI-MS, and database analysis revealed S100A10 as interacting protein. This result was confirmed by co-immunoprecipitation and immunocapturing. Using S100A10 as new bait, a specific interaction with S100A7 was detectable. PMID:16212425

  7. Fluorescence Studies of Protein Crystallization Interactions

    NASA Technical Reports Server (NTRS)

    Pusey, Marc L.; Smith, Lori; Forsythe, Elizabeth

    1999-01-01

    We are investigating protein-protein interactions in under- and over-saturated crystallization solution conditions using fluorescence methods. The use of fluorescence requires fluorescent derivatives where the probe does not markedly affect the crystal packing. A number of chicken egg white lysozyme (CEWL) derivatives have been prepared, with the probes covalently attached to one of two different sites on the protein molecule; the side chain carboxyl of ASP 101, within the active site cleft, and the N-terminal amine. The ASP 101 derivatives crystallize while the N-terminal amine derivatives do not. However, the N-terminal amine is part of the contact region between adjacent 43 helix chains, and blocking this site does would not interfere with formation of these structures in solution. Preliminary FRET data have been obtained at pH 4.6, 0.1M NaAc buffer, at 5 and 7% NaCl, 4 C, using the N-terminal bound pyrene acetic acid (PAA, Ex 340 nm, Em 376 nm) and ASP 101 bound Lucifer Yellow (LY, Ex 425 nm, Em 525 nm) probe combination. The corresponding Csat values are 0.471 and 0.362 mg/ml (approximately 3.3 and approximately 2.5 x 10 (exp 5) M respectively), and all experiments were carried out at approximately Csat or lower total protein concentration. The data at both salt concentrations show a consistent trend of decreasing fluorescence yield of the donor species (PAA) with increasing total protein concentration. This decrease is apparently more pronounced at 7% NaCl, consistent with the expected increased intermolecular interactions at higher salt concentrations (reflected in the lower solubility). The estimated average distance between protein molecules at 5 x 10 (exp 6) M is approximately 70 nm, well beyond the range where any FRET can be expected. The calculated RO, where 50% of the donor energy is transferred to the acceptor, for the PAA-CEWL * LY-CEWL system is 3.28 nm, based upon a PAA-CEWL quantum efficiency of 0.41.

  8. 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

  9. Modulation of nociceptive ion channels and receptors via protein-protein interactions: implications for pain relief

    PubMed Central

    Rouwette, Tom; Avenali, Luca; Sondermann, Julia; Narayanan, Pratibha; Gomez-Varela, David; Schmidt, Manuela

    2015-01-01

    In the last 2 decades biomedical research has provided great insights into the molecular signatures underlying painful conditions. However, chronic pain still imposes substantial challenges to researchers, clinicians and patients alike. Under pathological conditions, pain therapeutics often lack efficacy and exhibit only minimal safety profiles, which can be largely attributed to the targeting of molecules with key physiological functions throughout the body. In light of these difficulties, the identification of molecules and associated protein complexes specifically involved in chronic pain states is of paramount importance for designing selective interventions. Ion channels and receptors represent primary targets, as they critically shape nociceptive signaling from the periphery to the brain. Moreover, their function requires tight control, which is usually implemented by protein-protein interactions (PPIs). Indeed, manipulation of such PPIs entails the modulation of ion channel activity with widespread implications for influencing nociceptive signaling in a more specific way. In this review, we highlight recent advances in modulating ion channels and receptors via their PPI networks in the pursuit of relieving chronic pain. Moreover, we critically discuss the potential of targeting PPIs for developing novel pain therapies exhibiting higher efficacy and improved safety profiles. PMID:26039491

  10. Effect of thermal manipulation during embryogenesis on liver heat shock protein expression in chronic heat stressed colored broiler chickens.

    PubMed

    Vinoth, A; Thirunalasundari, T; Tharian, Jenny Anne; Shanmugam, M; Rajkumar, U

    2015-10-01

    Thermal manipulation during embryogenesis has been shown to improve thermo tolerance in broilers. Heat shock proteins are a family of proteins produced in response to variety of stress and protect cells from damage. The aim of this study was to evaluate the effect of thermal manipulation (TM) during embryogenesis on HSP gene and protein expression in the embryos and in chronic heat stressed 42nd day old chicks. On 15th day of incubation, fertile eggs from two breeds-Naked neck (NN) and Punjab Broiler-2 (PB-2) were randomly divided in to two groups, namely Control (C) eggs were incubated under standard incubation conditions and Thermal Conditioning (TC) eggs were exposed to higher incubation temperature (40.5°C) for 3h on 15th, 16th and 17th day of incubation. The chicks so obtained from each group were further subdivided and reared from 15th-42nd day as normal (N; 25±1°C, 70% RH) and heat exposed (HE; 35±1°C, 50% RH) resulting in four treatment groups (CN, CHE, TCN and TCHE). Embryos of two groups (C and TC) on 17th day and birds from four treatment groups on 42nd day were sacrificed. Liver was collected for analysis of gene expression by real-time PCR and protein expression by Western blot of Heat Shock Proteins (HSP 90 alpha, HSP 90 beta, HSP 70, HSP 60, HSP 27 and ubiquitin). The plasma collected on 42nd day was analyzed for biochemical parameters. Thermal challenging of embryos of both the breeds caused significant (P≤0.05) increase in all the HSPs gene and protein expression. The TCHE chicks had significantly (P≤0.05) lower HSPs gene and protein expressions and oxidative stress compared to CHE groups in both NN and PB-2. Based on these findings it can be concluded that TM during incubation provides adaptation to broiler chicks during chronic heat stress. PMID:26590469

  11. Prediction of Protein-Protein Interaction Sites Based on Naive Bayes Classifier

    PubMed Central

    Geng, Haijiang; Lu, Tao; Lin, Xiao; Liu, Yu; Yan, Fangrong

    2015-01-01

    Protein functions through interactions with other proteins and biomolecules and these interactions occur on the so-called interface residues of the protein sequences. Identifying interface residues makes us better understand the biological mechanism of protein interaction. Meanwhile, information about the interface residues contributes to the understanding of metabolic, signal transduction networks and indicates directions in drug designing. In recent years, researchers have focused on developing new computational methods for predicting protein interface residues. Here we creatively used a 181-dimension protein sequence feature vector as input to the Naive Bayes Classifier- (NBC-) based method to predict interaction sites in protein-protein complexes interaction. The prediction of interaction sites in protein interactions is regarded as an amino acid residue binary classification problem by applying NBC with protein sequence features. Independent test results suggested that Naive Bayes Classifier-based method with the protein sequence features as input vectors performed well. PMID:26697220

  12. Using visual cues of contact to improve interactive manipulation of virtual objects in industrial assembly/maintenance simulations.

    PubMed

    Sreng, Jean; Lécuyer, Anatole; Mégard, Christine; Andriot, Claude

    2006-01-01

    This paper describes a set of visual cues of contact designed to improve the interactive manipulation of virtual objects in industrial assembly/maintenance simulations. These visual cues display information of proximity, contact and effort between virtual objects when the user manipulates a part inside a digital mock-up. The set of visual cues encloses the apparition of glyphs (arrow, disk, or sphere) when the manipulated object is close or in contact with another part of the virtual environment. Light sources can also be added at the level of contact points. A filtering technique is proposed to decrease the number of glyphs displayed at the same time. Various effects--such as change in color, change in size, and deformation of shape- can be applied to the glyphs as a function of proximity with other objects or amplitude of the contact forces. A preliminary evaluation was conducted to gather the subjective preference of a group of participants during the simulation of an automotive assembly operation. The collected questionnaires showed that participants globally appreciated our visual cues of contact. The changes in color appeared to be preferred concerning the display of distances and proximity information. Size changes and deformation effects appeared to be preferred in terms of perception of contact forces between the parts. Last, light sources were selected to focus the attention of the user on the contact areas. PMID:17080829

  13. Protein-protein interactions in plant mitogen-activated protein kinase cascades.

    PubMed

    Zhang, Tong; Chen, Sixue; Harmon, Alice C

    2016-02-01

    Mitogen-activated protein kinases (MAPKs) form tightly controlled signaling cascades that play essential roles in plant growth, development, and defense. However, the molecular mechanisms underlying MAPK cascades are still elusive, due largely to our poor understanding of how they relay the signals. Extensive effort has been devoted to characterization of MAPK-substrate interactions to illustrate phosphorylation-based signaling. The diverse MAPK substrates identified also shed light on how spatiotemporal-specific protein-protein interactions function in distinct MAPK cascade-mediated biological processes. This review surveys various technologies used for characterizing MAPK-substrate interactions and presents case studies of MPK4 and MPK6, highlighting the multiple functions of MAPKs. Mass spectrometry-based approaches in identifying MAPK-interacting proteins are emphasized due to their increasing utility and effectiveness. The potential for using MAPKs and their substrates in enhancing plant stress tolerance is also discussed. PMID:26646897

  14. The Foundations of Protein-Ligand Interaction

    NASA Astrophysics Data System (ADS)

    Klebe, Gerhard

    For the specific design of a drug we must first answer the question: How does a drug achieve its activity? An active ingredient must, in order to develop its action, bind to a particular target molecule in the body. Usually this is a protein, but also nucleic acids in the form of RNA and DNA can be target structures for active agents. The most important condition for binding is at first that the active agent exhibits the correct size and shape in order to optimally fit into a cavity exposed to the surface of the protein, the "bindingpocket". It is further necessary for the surface properties of the ligand and protein to be mutually compatible to form specific interactions. In 1894 Emil Fischer compared the exact fit of a substrate for the catalytic centre of an enzyme with the picture of a "lock-and-key". Paul Ehrlich coined in 1913 "Corpora non agunt nisi fixata", literally "bodies do not work when they are not bound". He wanted to imply that active agents that are meant to kill bacteria or parasites must be "fixed" by them, i.e. linked to their structures. Both concepts form the starting point for any rational concept in the development of active pharmaceutical ingredients. In many respects they still apply today. A drug must, after being administered, reach its target and interact with a biological macromolecule. Specific agents have a large affinity and sufficient selectivity to bind to the macromolecule's active site. This is the only way they can develop the desired biological activity without side-effects.

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

    PubMed

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

    2015-01-01

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

  16. Manipulating the glycosylation pathway in bacterial and lower eukaryotes for production of therapeutic proteins.

    PubMed

    Anyaogu, Diana Chinyere; Mortensen, Uffe Hasbro

    2015-12-01

    The medical use of pharmaceutical proteins is rapidly increasing and cheap, fast and efficient production is therefore attractive. Microbial production hosts are promising candidates for development and production of pharmaceutical proteins. However, as most therapeutic proteins are secreted proteins, they are frequently N-glycosylated. This hampers production in microbes as these hosts glycosylate proteins differently. The resulting products may therefore be immunogenic, unstable and show reduced efficacy. Recently, successful glycoengineering of microbes has demonstrated that it is possible to produce proteins with humanlike glycan structures setting the stage for production of pharmaceutical proteins in bacteria, yeasts and algae. PMID:26340101

  17. Methods for Mapping of Interaction Networks Involving Membrane Proteins

    SciTech Connect

    Hooker, Brian S.; Bigelow, Diana J.; Lin, Chiann Tso

    2007-11-23

    Numerous approaches have been taken to study protein interactions, such as tagged protein complex isolation followed by mass spectrometry, yeast two-hybrid methods, fluorescence resonance energy transfer, surface plasmon resonance, site-directed mutagenesis, and crystallography. Membrane protein interactions pose significant challenges due to the need to solubilize membranes without disrupting protein-protein interactions. Traditionally, analysis of isolated protein complexes by high-resolution 2D gel electrophoresis has been the main method used to obtain an overall picture of proteome constituents and interactions. However, this method is time consuming, labor intensive, detects only abundant proteins and is not suitable for the coverage required to elucidate large interaction networks. In this review, we discuss the application of various methods to elucidate interactions involving membrane proteins. These techniques include methods for the direct isolation of single complexes or interactors as well as methods for characterization of entire subcellular and cellular interactomes.

  18. Essential protein identification based on essential protein-protein interaction prediction by Integrated Edge Weights.

    PubMed

    Jiang, Yuexu; Wang, Yan; Pang, Wei; Chen, Liang; Sun, Huiyan; Liang, Yanchun; Blanzieri, Enrico

    2015-07-15

    Essential proteins play a crucial role in cellular survival and development process. Experimentally, essential proteins are identified by gene knockouts or RNA interference, which are expensive and often fatal to the target organisms. Regarding this, an alternative yet important approach to essential protein identification is through computational prediction. Existing computational methods predict essential proteins based on their relative densities in a protein-protein interaction (PPI) network. Degree, betweenness, and other appropriate criteria are often used to measure the relative density. However, no matter what criterion is used, a protein is actually ordered by the attributes of this protein per se. In this research, we presented a novel computational method, Integrated Edge Weights (IEW), to first rank protein-protein interactions by integrating their edge weights, and then identified sub PPI networks consisting of those highly-ranked edges, and finally regarded the nodes in these sub networks as essential proteins. We evaluated IEW on three model organisms: Saccharomyces cerevisiae (S. cerevisiae), Escherichia coli (E. coli), and Caenorhabditis elegans (C. elegans). The experimental results showed that IEW achieved better performance than the state-of-the-art methods in terms of precision-recall and Jackknife measures. We had also demonstrated that IEW is a robust and effective method, which can retrieve biologically significant modules by its highly-ranked protein-protein interactions for S. cerevisiae, E. coli, and C. elegans. We believe that, with sufficient data provided, IEW can be used to any other organisms' essential protein identification. A website about IEW can be accessed from http://digbio.missouri.edu/IEW/index.html. PMID:25892709

  19. Protein-protein interactions of PDE4 family members - Functions, interactions and therapeutic value.

    PubMed

    Klussmann, Enno

    2016-07-01

    The second messenger cyclic adenosine monophosphate (cAMP) is ubiquitous and directs a plethora of functions in all cells. Although theoretically freely diffusible through the cell from the site of its synthesis it is not evenly distributed. It rather is shaped into gradients and these gradients are established by phospodiesterases (PDEs), the only enzymes that hydrolyse cAMP and thereby terminate cAMP signalling upstream of cAMP's effector systems. Miles D. Houslay has devoted most of his scientific life highly successfully to a particular family of PDEs, the PDE4 family. The family is encoded by four genes and gives rise to around 20 enzymes, all with different functions. M. Houslay has discovered many of these functions and realised early on that PDE4 family enzymes are attractive drug targets in a variety of human diseases, but not their catalytic activity as that is encoded in conserved domains in all family members. He postulated that targeting the intracellular location would provide the specificity that modern innovative drugs require to improve disease conditions with fewer side effects than conventional drugs. Due to the wealth of M. Houslay's work, this article can only summarize some of his discoveries and, therefore, focuses on protein-protein interactions of PDE4. The aim is to discuss functions of selected protein-protein interactions and peptide spot technology, which M. Houslay introduced into the PDE4 field for identifying interacting domains. The therapeutic potential of PDE4 interactions will also be discussed. PMID:26498857

  20. Intricate protein-protein interactions in the cyanobacterial circadian clock.

    PubMed

    Egli, Martin

    2014-08-01

    The cyanobacterial circadian clock consists of a post-translational oscillator (PTO) and a PTO-dependent transcription-translation feedback loop (TTFL). The PTO can be reconstituted in vitro with the KaiA, KaiB, and KaiC proteins, enabling detailed biochemical and biophysical investigations. Both the CI and the CII halves of the KaiC hexamer harbor ATPases, but only the C-terminal CII ring exhibits kinase and phospho-transferase activities. KaiA stimulates the kinase and KaiB associates with KaiC during the dephosphorylation phase and sequesters KaiA. Recent research has led to conflicting models of the KaiB-KaiC interaction, precluding a clear understanding of KaiB function and KaiABC clock mechanism. PMID:24936066

  1. Intricate Protein-Protein Interactions in the Cyanobacterial Circadian Clock*

    PubMed Central

    Egli, Martin

    2014-01-01

    The cyanobacterial circadian clock consists of a post-translational oscillator (PTO) and a PTO-dependent transcription-translation feedback loop (TTFL). The PTO can be reconstituted in vitro with the KaiA, KaiB, and KaiC proteins, enabling detailed biochemical and biophysical investigations. Both the CI and the CII halves of the KaiC hexamer harbor ATPases, but only the C-terminal CII ring exhibits kinase and phospho-transferase activities. KaiA stimulates the kinase and KaiB associates with KaiC during the dephosphorylation phase and sequesters KaiA. Recent research has led to conflicting models of the KaiB-KaiC interaction, precluding a clear understanding of KaiB function and KaiABC clock mechanism. PMID:24936066

  2. A logical molecular circuit for programmable and autonomous regulation of protein activity using DNA aptamer-protein interactions

    PubMed Central

    Han, Da; Zhu, Zhi; Wu, Cuichen; Peng, Lu; Zhou, Leiji; Gulbakan, Basri; Zhu, Guizhi; Williams, Kathryn R.; Tan, Weihong

    2013-01-01

    Researchers increasingly envision an important role for artificial biochemical circuits in biological engineering, much like electrical circuits in electrical engineering. Similar to electrical circuits, which control electromechanical devices, biochemical circuits could be utilized as a type of servomechanism to control nanodevices in vitro, monitor chemical reactions in situ, or regulate gene expressions in vivo.1 As a consequence of their relative robustness and potential applicability for controlling a wide range of in vitro chemistries, synthetic cell-free biochemical circuits promise to be useful in manipulating the functions of biological molecules. Here we describe the first logical circuit based on DNA-protein interactions with accurate threshold control, enabling autonomous, self-sustained and programmable manipulation of protein activity in vitro. Similar circuits made previously were based primarily on DNA hybridization and strand displacement reactions. This new design uses the diverse nucleic acid interactions with proteins. The circuit can precisely sense the local enzymatic environment, such as the concentration of thrombin, and when it is excessively high, a coagulation inhibitor is automatically released by a concentration-adjusted circuit module. To demonstrate the programmable and autonomous modulation, a molecular circuit with different threshold concentrations of thrombin was tested as a proof of principle. In the future, owing to tunable regulation, design modularity and target specificity, this prototype could lead to the development of novel DNA biochemical circuits to control the delivery of aptamer-based drugs in smart and personalized medicine, providing a more efficient and safer therapeutic strategy. PMID:23194304

  3. A logical molecular circuit for programmable and autonomous regulation of protein activity using DNA aptamer-protein interactions.

    PubMed

    Han, Da; Zhu, Zhi; Wu, Cuichen; Peng, Lu; Zhou, Leiji; Gulbakan, Basri; Zhu, Guizhi; Williams, Kathryn R; Tan, Weihong

    2012-12-26

    Researchers increasingly envision an important role for artificial biochemical circuits in biological engineering, much like electrical circuits in electrical engineering. Similar to electrical circuits, which control electromechanical devices, biochemical circuits could be utilized as a type of servomechanism to control nanodevices in vitro, monitor chemical reactions in situ, or regulate gene expressions in vivo. (1) As a consequence of their relative robustness and potential applicability for controlling a wide range of in vitro chemistries, synthetic cell-free biochemical circuits promise to be useful in manipulating the functions of biological molecules. Here, we describe the first logical circuit based on DNA-protein interactions with accurate threshold control, enabling autonomous, self-sustained and programmable manipulation of protein activity in vitro. Similar circuits made previously were based primarily on DNA hybridization and strand displacement reactions. This new design uses the diverse nucleic acid interactions with proteins. The circuit can precisely sense the local enzymatic environment, such as the concentration of thrombin, and when it is excessively high, a coagulation inhibitor is automatically released by a concentration-adjusted circuit module. To demonstrate the programmable and autonomous modulation, a molecular circuit with different threshold concentrations of thrombin was tested as a proof of principle. In the future, owing to tunable regulation, design modularity and target specificity, this prototype could lead to the development of novel DNA biochemical circuits to control the delivery of aptamer-based drugs in smart and personalized medicine, providing a more efficient and safer therapeutic strategy. PMID:23194304

  4. Specificity and non-specificity in RNA–protein interactions

    PubMed Central

    Jankowsky, Eckhard; Harris, Michael E.

    2016-01-01

    Gene expression is regulated by complex networks of interactions between RNAs and proteins. Proteins that interact with RNA have been traditionally viewed as either specific or non-specific; specific proteins interact preferentially with defined RNA sequence or structure motifs, whereas non-specific proteins interact with RNA sites devoid of such characteristics. Recent studies indicate that the binary “specific vs. non-specific” classification is insufficient to describe the full spectrum of RNA–protein interactions. Here, we review new methods that enable quantitative measurements of protein binding to large numbers of RNA variants, and the concepts aimed as describing resulting binding spectra: affinity distributions, comprehensive binding models and free energy landscapes. We discuss how these new methodologies and associated concepts enable work towards inclusive, quantitative models for specific and non-specific RNA–protein interactions. PMID:26285679

  5. Methods for the analysis of protein-chromatin interactions.

    PubMed

    Brickwood, Sarah J; Myers, Fiona A; Chandler, Simon P

    2002-01-01

    The analysis of protein interactions with chromatin is vital for the understanding of DNA sequence recognition in vivo. Chromatin binding requires the interaction of proteins with DNA lying on the macromolecular protein surface of nucleosomes, a situation that can alter factor binding characteristics substantially when compared with naked DNA. It is therefore important to study these protein-DNA interactions in the context of a chromatin substrate, the more physiologically relevant binding situation. In this article we review techniques used in the investigation of protein interactions with defined nucleosomal templates. PMID:11876294

  6. The Use of Interactive Raster Graphics in the Display and Manipulation of Multidimensional Data

    NASA Technical Reports Server (NTRS)

    Anderson, D. C.

    1981-01-01

    Techniques for the review, display, and manipulation of multidimensional data are developed and described. Multidimensional data is meant in this context to describe scalar data associated with a three dimensional geometry or otherwise too complex to be well represented by traditional graphs. Raster graphics techniques are used to display a shaded image of a three dimensional geometry. The use of color to represent scalar data associated with the geometries in shaded images is explored. Distinct hues are associated with discrete data ranges, thus emulating the traditional representation of data with isarithms, or lines of constant numerical value. Data ranges are alternatively associated with a continuous spectrum of hues to show subtler data trends. The application of raster graphics techniques to the display of bivariate functions is explored.

  7. Interaction and localization diversities of global and local hubs in human protein-protein interaction networks.

    PubMed

    Kiran, M; Nagarajaram, H A

    2016-08-16

    Hubs, the highly connected nodes in protein-protein interaction networks (PPINs), are associated with several characteristic properties and are known to perform vital roles in cells. We defined two classes of hubs, global (housekeeping) and local (tissue-specific) hubs. These two categories of hubs are distinct from each other with respect to their abundance, structure and function. However, how distinct are the spatial expression pattern and other characteristics of their interacting partners is still not known. Our investigations revealed that the partners of the local hubs compared with those of global hubs are conserved across the tissues in which they are expressed. Partners of local hubs show diverse subcellular localizations as compared with the partners of global hubs. We examined the nature of interacting domains in both categories of hubs and found that they are promiscuous in global hubs but not so in local hubs. Deletion of some of the local and global hubs has an impact on the characteristic path length of the network indicating that those hubs are inter-modular in nature. Our present study has, therefore, shed further light on the characteristic features of the local and global hubs in human PPIN. This knowledge of different topological aspects of hubs with regard to their types and subtypes is essential as it helps in better understanding of roles of hub proteins in various cellular processes under various conditions including those caused by host-pathogen interactions and therefore useful in prioritizing targets for drug design and repositioning. PMID:27400769

  8. Evolution of protein interactions: from interactomes to interfaces.

    PubMed

    Andreani, Jessica; Guerois, Raphael

    2014-07-15

    Protein-protein interactions lie at the heart of most cellular processes. Many experimental and computational studies aim to deepen our understanding of these interactions and improve our capacity to predict them. In this respect, the evolutionary perspective is most interesting, since the preservation of structure and function puts constraints on the evolution of proteins and their interactions. However, uncovering these constraints remains a challenge, and the description and detection of evolutionary signals in protein-protein interactions is currently a very active field of research. Here, we review recent works dissecting the mechanisms of protein-protein interaction evolution and exploring how to use evolutionary information to predict interactions, both at the global level of the interactome and at the detailed level of protein-protein interfaces. We first present to what extent protein-protein interactions are found to be conserved within interactomes and which properties can influence their conservation. We then discuss the evolutionary and co-evolutionary pressures applied on protein-protein interfaces. Finally, we describe how the computational prediction of interfaces can benefit from evolutionary inputs. PMID:24853495

  9. Non-photic manipulations induce expression of Fos protein in the suprachiasmatic nucleus and intergeniculate leaflet in the rat.

    PubMed

    Edelstein, K; Amir, S

    1995-09-01

    Expression of Fos protein in the suprachiasmatic nucleus (SCN) and intergeniculate leaflet (IGL) is considered a cellular correlate of light-induced phase-shift of circadian rhythms in rodents. Non-photic stimuli also induce phase shifts, but their effects on Fos expression have not been established. We examined induction of Fos protein in SCN and IGL regions, in response to cage change, intraperitoneal saline injection, and restraint stress. Fos immunoreactivity was observed in SCN and IGL regions, with greater expression observed in IGL during the light phase of the light-dark cycle. Results suggest that cells in SCN and IGL respond to several types of non-photic manipulations and that expression of Fos in these regions is not light-specific. PMID:8535846

  10. Probing calmodulin protein-protein interactions using high-content protein arrays.

    PubMed

    O'Connell, David J; Bauer, Mikael; Linse, Sara; Cahill, Dolores J

    2011-01-01

    The calcium ion (Ca(2+)) is a ubiquitous second messenger that is crucial for the regulation of a wide variety of cellular processes. The diverse transient signals transduced by Ca(2+) are mediated by intracellular -Ca(2+)-binding proteins. Calcium ions shuttle into and out of the cytosol, transported across membranes by channels, exchangers, and pumps that regulate flux across the ER, mitochondrial and plasma membranes. Calcium regulates both rapid events, such as cytoskeleton remodelling or release of vesicle contents, and slower ones, such as transcriptional changes. Moreover, sustained cytosolic calcium elevations can lead to unwanted cellular activation or apoptosis. Calmodulin represents the most significant of the Ca(2+)-binding proteins and is an essential regulator of intracellular processes in response to extracellular stimuli mediated by a rise in Ca(2+) ion concentration. To profile novel protein-protein interactions that calmodulin participates in, we probed a high-content recombinant human protein array with fluorophore-labelled calmodulin in the presence of Ca(2+). This protein array contains 37,200 redundant proteins, incorporating over 10,000 unique human proteins expressed from a human brain cDNA library. We describe the identification of a high affinity interaction between calmodulin and the single-pass transmembrane proteins STIM1 and STIM2 that localise to the ER. Translocation of STIM1 and STIM2 from the endoplasmic reticulum to the plasma membrane is a key step in store operated calcium entry in the cell. PMID:21901608

  11. Collective prediction of protein functions from protein-protein interaction networks

    PubMed Central

    2014-01-01

    Background Automated assignment of functions to unknown proteins is one of the most important task in computational biology. The development of experimental methods for genome scale analysis of molecular interaction networks offers new ways to infer protein function from protein-protein interaction (PPI) network data. Existing techniques for collective classification (CC) usually increase accuracy for network data, wherein instances are interlinked with each other, using a large amount of labeled data for training. However, the labeled data are time-consuming and expensive to obtain. On the other hand, one can easily obtain large amount of unlabeled data. Thus, more sophisticated methods are needed to exploit the unlabeled data to increase prediction accuracy for protein function prediction. Results In this paper, we propose an effective Markov chain based CC algorithm (ICAM) to tackle the label deficiency problem in CC for interrelated proteins from PPI networks. Our idea is to model the problem using two distinct Markov chain classifiers to make separate predictions with regard to attribute features from protein data and relational features from relational information. The ICAM learning algorithm combines the results of the two classifiers to compute the ranks of labels to indicate the importance of a set of labels to an instance, and uses an ICA framework to iteratively refine the learning models for improving performance of protein function prediction from PPI networks in the paucity of labeled data. Conclusion Experimental results on the real-world Yeast protein-protein interaction datasets show that our proposed ICAM method is better than the other ICA-type methods given limited labeled training data. This approach can serve as a valuable tool for the study of protein function prediction from PPI networks. PMID:24564855

  12. Alterations of social interaction through genetic and environmental manipulation of the 22q11.2 gene Sept5 in the mouse brain.

    PubMed

    Harper, Kathryn M; Hiramoto, Takeshi; Tanigaki, Kenji; Kang, Gina; Suzuki, Go; Trimble, William; Hiroi, Noboru

    2012-08-01

    Social behavior dysfunction is a symptomatic element of schizophrenia and autism spectrum disorder (ASD). Although altered activities in numerous brain regions are associated with defective social cognition and perception, the causative relationship between these altered activities and social cognition and perception-and their genetic underpinnings-are not known in humans. To address these issues, we took advantage of the link between hemizygous deletion of human chromosome 22q11.2 and high rates of social behavior dysfunction, schizophrenia and ASD. We genetically manipulated Sept5, a 22q11.2 gene, and evaluated its role in social interaction in mice. Sept5 deficiency, against a high degree of homogeneity in a congenic genetic background, selectively impaired active affiliative social interaction in mice. Conversely, virally guided overexpression of Sept5 in the hippocampus or, to a lesser extent, the amygdala elevated levels of active affiliative social interaction in C57BL/6J mice. Congenic knockout mice and mice overexpressing Sept5 in the hippocampus or amygdala were indistinguishable from control mice in novelty and olfactory responses, anxiety or motor activity. Moreover, post-weaning individual housing, an environmental condition designed to reduce stress in male mice, selectively raised levels of Sept5 protein in the amygdala and increased active affiliative social interaction in C57BL/6J mice. These findings identify this 22q11.2 gene in the hippocampus and amygdala as a determinant of social interaction and suggest that defective social interaction seen in 22q11.2-associated schizophrenia and ASD can be genetically and environmentally modified by altering this 22q11.2 gene. PMID:22589251

  13. Ensemble learning prediction of protein-protein interactions using proteins functional annotations.

    PubMed

    Saha, Indrajit; Zubek, Julian; Klingström, Tomas; Forsberg, Simon; Wikander, Johan; Kierczak, Marcin; Maulik, Ujjwal; Plewczynski, Dariusz

    2014-04-01

    Protein-protein interactions are important for the majority of biological processes. A significant number of computational methods have been developed to predict protein-protein interactions using protein sequence, structural and genomic data. Vast experimental data is publicly available on the Internet, but it is scattered across numerous databases. This fact motivated us to create and evaluate new high-throughput datasets of interacting proteins. We extracted interaction data from DIP, MINT, BioGRID and IntAct databases. Then we constructed descriptive features for machine learning purposes based on data from Gene Ontology and DOMINE. Thereafter, four well-established machine learning methods: Support Vector Machine, Random Forest, Decision Tree and Naïve Bayes, were used on these datasets to build an Ensemble Learning method based on majority voting. In cross-validation experiment, sensitivity exceeded 80% and classification/prediction accuracy reached 90% for the Ensemble Learning method. We extended the experiment to a bigger and more realistic dataset maintaining sensitivity over 70%. These results confirmed that our datasets are suitable for performing PPI prediction and Ensemble Learning method is well suited for this task. Both the processed PPI datasets and the software are available at . PMID:24469380

  14. Protein tertiary structure recognition using optimized Hamiltonians with local interactions.

    PubMed Central

    Goldstein, R A; Luthey-Schulten, Z A; Wolynes, P G

    1992-01-01

    Protein folding codes embodying local interactions including surface and secondary structure propensities and residue-residue contacts are optimized for a set of training proteins by using spin-glass theory. A screening method based on these codes correctly matches the structure of a set of test proteins with proteins of similar topology with 100% accuracy, even with limited sequence similarity between the test proteins and the structural homologs and the absence of any structurally similar proteins in the training set. PMID:1409599

  15. Predicting the binding patterns of hub proteins: a study using yeast protein interaction networks

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Protein-protein interactions are critical to elucidating the role played by individual proteins in important biological pathways. Such networks are typically constructed using high throughput techniques (e.g., Yeast-2-Hybrid experiments). Of particular interest are hub proteins that can interact wit...

  16. A Laboratory-Intensive Course on the Experimental Study of Protein-Protein Interactions

    ERIC Educational Resources Information Center

    Witherow, D. Scott; Carson, Sue

    2011-01-01

    The study of protein-protein interactions is important to scientists in a wide range of disciplines. We present here the assessment of a lab-intensive course that teaches students techniques used to identify and further study protein-protein interactions. One of the unique elements of the course is that students perform a yeast two-hybrid screen…

  17. 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

  18. Studying Protein-Protein Interactions in Budding Yeast Using Co-immunoprecipitation.

    PubMed

    Foltman, Magdalena; Sanchez-Diaz, Alberto

    2016-01-01

    Understanding protein-protein interactions and the architecture of protein complexes in which they work is essential to identify their biological role. Protein co-immunoprecipitation (co-IP) is an invaluable technique used in biochemistry allowing the identification of protein interactors. Here, we describe in detail an immunoaffinity purification protocol as a one-step or two-step immunoprecipitation from budding yeast Saccharomyces cerevisiae cells to subsequently detect interactions between proteins involved in the same biological process. PMID:26519317

  19. Manipulation of the resonance interaction in Mach-Zehnder-Fano interferometers

    SciTech Connect

    Xu Yi; Miroshnichenko, Andrey E.

    2011-09-15

    We study tunable interaction of the resonances in the Mach-Zehnder-Fano interferometers (MZFIs). A discrete Mach-Zehnder interferometer (MZI) with balanced arms supports bound states in the continuum. We demonstrate that doping an impurity in conventional MZIs gives rise to robust high-Q Fano resonances with asymmetric line shapes. By means of the modified Fano-Anderson model and the scattering-matrix approach, we show that the transmission and the intensity spectra of the whole system are very sensitive to both the location and the strength of the impurity. The side-coupled Fano defects induce an interaction with different eigenmodes of the pure MZI loop. We explore this interaction by tuning the parameters of the Fano defects. The observed resonance interaction can be attributed to the Fano-Feshbach resonance. We further provide with a particular physical example of photonic crystal circuit the applicability of our concept.

  20. Interactive manipulation of photogrammetric data with the Zeiss-AS-programm system for minicomputers

    NASA Astrophysics Data System (ADS)

    Hobbie, D.

    1980-03-01

    The company of Carl Zeiss, Oberkochen, presents the AS Program System for HP 1000 minicomputer, which allows interactive operation during digital measurements with on line connected mono- and stereocomparators as well as stereoplotters. Operation is based on the menu technique. By comparison with the off line technique, interactive operation reduces transfer errors, allows direct checking of measured data by computation and provides direct access to further programs for processing or on files.

  1. In Vivo Application of Photocleavable Protein Interaction Reporter Technology

    PubMed Central

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

    2012-01-01

    Summary In vivo protein structures and protein-protein interactions are critical to the function of proteins in biological systems. As a complementary approach to traditional protein interaction identification methods, cross-linking strategies are beginning to provide additional data on protein and protein complex topological features. Previously, photocleavable protein interaction reporter (pcPIR) technology was demonstrated by cross-linking pure proteins and protein complexes and the use of ultraviolet light to cleave or release cross-linked peptides to enable identification. In the present report, the pcPIR strategy is applied to E. coli cells and in vivo protein interactions and topologies are measured. More than 1600 labeled peptides from E. coli were identified, indicating many protein sites react with pcPIR in vivo. From those labeled sites, 53 in vivo inter-cross-linked peptide pairs were identified and manually validated. Approximately half of the interactions have been reported using other techniques, although detailed structures exist for very few. Three proteins or protein complexes with detailed crystallography structures are compared to the cross-linking results obtained from in vivo application of pcPIR technology. PMID:22168182

  2. Interactions of proteins in gels, solutions and on surfaces

    NASA Astrophysics Data System (ADS)

    Ramasamy, Radha Perumal

    2006-12-01

    The study of protein interaction, identification and separation has applications in various fields relating to Biotechnology. In this research these aspects were investigated. The proteins albumin, casein, poly-L-lysine were studied. FITC and TRITC were used to fluorescently tag the proteins. Confocal microscopy was used to image the interaction of proteins. The migration of fluorescently tagged protein-salt aggregates on solid surfaces during electrophoresis was investigated using Confocal microscopy. The secondary structural modifications of proteins in solutions were investigated using FTIR micro spectroscopic imaging. The size of the colloids formed due to protein-protein interactions as a function of the protein concentrations were studied using DLS and their charges were found using zeta potential measurements. Based on DL.S and zeta potential measurements, a model is proposed for interactions of oppositely charged proteins. The nature of interaction was found using UV - Visual spectroscopy. It was found that oppositely charged proteins formed ionic bonds. It was also found that FITC molecule influenced the surface charge of albumin more than TRITC molecule. The effects of the influence of cell geometries upon Electro Osmotic Flow (EOF) were studied using neutrally charged fluorescent Polystyrene beads. Results showed that tagging proteins with fluorescent molecules influenced their mobility and interactions with other proteins. However no secondary structural modifications of the proteins were observed when oppositely charged proteins interacted. It was also observed that electrostatic interactions made oppositely charged proteins form large aggregates. The EOF was found to be dependent upon the ionic strength of the buffer, conductivity of the solid surfaces, distance from the surface and position of the electrodes in the electrophoretic cell.

  3. Measuring protein-protein and protein-nucleic Acid interactions by biolayer interferometry.

    PubMed

    Sultana, Azmiri; Lee, Jeffrey E

    2015-01-01

    Biolayer interferometry (BLI) is a simple, optical dip-and-read system useful for measuring interactions between proteins, peptides, nucleic acids, small molecules, and/or lipids in real time. In BLI, a biomolecular bait is immobilized on a matrix at the tip of a fiber-optic sensor. The binding between the immobilized ligand and another molecule in an analyte solution produces a change in optical thickness at the tip and results in a wavelength shift proportional to binding. BLI provides direct binding affinities and rates of association and dissociation. This unit describes an efficient approach using streptavidin-based BLI to analyze DNA-protein and protein-protein interactions. A quantitative set of equilibrium binding affinities (K(d)) and rates of association and dissociation (k(a)/k(d)) can be measured in minutes using nanomole quantities of sample. PMID:25640894

  4. Modularity in the evolution of yeast protein interaction network

    PubMed Central

    Ogishima, Soichi; Tanaka, Hiroshi; Nakaya, Jun

    2015-01-01

    Protein interaction networks are known to exhibit remarkable structures: scale-free and small-world and modular structures. To explain the evolutionary processes of protein interaction networks possessing scale-free and small-world structures, preferential attachment and duplication-divergence models have been proposed as mathematical models. Protein interaction networks are also known to exhibit another remarkable structural characteristic, modular structure. How the protein interaction networks became to exhibit modularity in their evolution? Here, we propose a hypothesis of modularity in the evolution of yeast protein interaction network based on molecular evolutionary evidence. We assigned yeast proteins into six evolutionary ages by constructing a phylogenetic profile. We found that all the almost half of hub proteins are evolutionarily new. Examining the evolutionary processes of protein complexes, functional modules and topological modules, we also found that member proteins of these modules tend to appear in one or two evolutionary ages. Moreover, proteins in protein complexes and topological modules show significantly low evolutionary rates than those not in these modules. Our results suggest a hypothesis of modularity in the evolution of yeast protein interaction network as systems evolution. PMID:25914446

  5. Extreme multifunctional proteins identified from a human protein interaction network

    PubMed Central

    Chapple, Charles E.; Robisson, Benoit; Spinelli, Lionel; Guien, Céline; Becker, Emmanuelle; Brun, Christine

    2015-01-01

    Moonlighting proteins are a subclass of multifunctional proteins whose functions are unrelated. Although they may play important roles in cells, there has been no large-scale method to identify them, nor any effort to characterize them as a group. Here, we propose the first method for the identification of ‘extreme multifunctional' proteins from an interactome as a first step to characterize moonlighting proteins. By combining network topological information with protein annotations, we identify 430 extreme multifunctional proteins (3% of the human interactome). We show that the candidates form a distinct sub-group of proteins, characterized by specific features, which form a signature of extreme multifunctionality. Overall, extreme multifunctional proteins are enriched in linear motifs and less intrinsically disordered than network hubs. We also provide MoonDB, a database containing information on all the candidates identified in the analysis and a set of manually curated human moonlighting proteins. PMID:26054620

  6. Course 1: Physics of Protein-DNA Interaction

    NASA Astrophysics Data System (ADS)

    Bruinsma, R. F.

    1 Introduction 1.1 The central dogma and bacterial gene expression 1.2 Molecular structure 2 Thermodynamics and kinetics of repressor-DNA interaction 2.1 Thermodynamics and the lac repressor 2.2 Kinetics of repressor-DNA interaction 3 DNA deformability and protein-DNA interaction 3.1 Introduction 3.2 The worm-like chain 3.3 The RST model 4 Electrostatics in water and protein-DNA interaction 4.1 Macro-ions and aqueous electrostatics 4.2 The primitive model 4.3 Manning condensation 4.4 Counter-ion release and non-specific protein-DNA interaction

  7. Folding superfunnel to describe cooperative folding of interacting proteins.

    PubMed

    Smeller, László

    2016-07-01

    This paper proposes a generalization of the well-known folding funnel concept of proteins. In the funnel model the polypeptide chain is treated as an individual object not interacting with other proteins. Since biological systems are considerably crowded, protein-protein interaction is a fundamental feature during the life cycle of proteins. The folding superfunnel proposed here describes the folding process of interacting proteins in various situations. The first example discussed is the folding of the freshly synthesized protein with the aid of chaperones. Another important aspect of protein-protein interactions is the folding of the recently characterized intrinsically disordered proteins, where binding to target proteins plays a crucial role in the completion of the folding process. The third scenario where the folding superfunnel is used is the formation of aggregates from destabilized proteins, which is an important factor in case of several conformational diseases. The folding superfunnel constructed here with the minimal assumption about the interaction potential explains all three cases mentioned above. Proteins 2016; 84:1009-1016. © 2016 Wiley Periodicals, Inc. PMID:27090200

  8. An interactive graphics program to retrieve, display, compare, manipulate, curve fit, difference and cross plot wind tunnel data

    NASA Technical Reports Server (NTRS)

    Elliott, R. D.; Werner, N. M.; Baker, W. M.

    1975-01-01

    The Aerodynamic Data Analysis and Integration System (ADAIS), developed as a highly interactive computer graphics program capable of manipulating large quantities of data such that addressable elements of a data base can be called up for graphic display, compared, curve fit, stored, retrieved, differenced, etc., was described. The general nature of the system is evidenced by the fact that limited usage has already occurred with data bases consisting of thermodynamic, basic loads, and flight dynamics data. Productivity using ADAIS of five times that for conventional manual methods of wind tunnel data analysis is routinely achieved. In wind tunnel data analysis, data from one or more runs of a particular test may be called up and displayed along with data from one or more runs of a different test. Curves may be faired through the data points by any of four methods, including cubic spline and least squares polynomial fit up to seventh order.

  9. Primary reaction control system/remote manipulator system interaction with loaded arm. Space shuttle engineering and operations support

    NASA Technical Reports Server (NTRS)

    Taylor, E. C.; Davis, J. D.

    1978-01-01

    A study of the interaction between the orbiter primary reaction control system (PRCS) and the remote manipulator system (RMS) with a loaded arm is documented. This analysis was performed with the Payload Deployment and Retrieval Systems Simulation (PDRSS) program with the passive arm bending option. The passive-arm model simulates the arm as massless elastic links with locked joints. The study was divided into two parts. The first part was the evaluation of the response of the arm to step inputs (i.e. constant jet torques) about each of the orbiter body axes. The second part of the study was the evaluation of the response of the arm to minimum impulse primary RCS jet firings with both single pulse and pulse train inputs.

  10. A Proteomic approach to discover and compare interacting partners of Papillomavirus E2 proteins from diverse phylogenetic groups

    PubMed Central

    Jang, Moon Kyoo; Anderson, D. Eric; van Doorslaer, Koenraad; McBride, Alison A.

    2015-01-01

    Papillomaviruses are a very successful group of viruses that replicate persistently in localized regions of the stratified epithelium of their specific host. Infection results in pathologies ranging from asymptomatic infection, benign warts, to malignant carcinomas. Despite this diversity, papillomavirus genomes are small (7-8 kbp) and contain at most eight genes. To sustain the complex papillomaviral life cycle, each viral protein has multiple functions and interacts with and manipulates a plethora of cellular proteins. In this study, we use tandem affinity purification and mass spectrometry to identify host factors that interact with eleven different papillomavirus E2 proteins from diverse phylogenetic groups. The E2 proteins function in viral transcription and replication and correspondingly interact with host proteins involved in transcription, chromatin remodeling and modification, replication and RNA processing. PMID:25758368

  11. A proteomic approach to discover and compare interacting partners of papillomavirus E2 proteins from diverse phylogenetic groups.

    PubMed

    Jang, Moon Kyoo; Anderson, D Eric; van Doorslaer, Koenraad; McBride, Alison A

    2015-06-01

    Papillomaviruses are a very successful group of viruses that replicate persistently in localized regions of the stratified epithelium of their specific host. Infection results in pathologies ranging from asymptomatic infection, benign warts, to malignant carcinomas. Despite this diversity, papillomavirus genomes are small (7-8 kbp) and contain at most eight genes. To sustain the complex papillomaviral life cycle, each viral protein has multiple functions and interacts with and manipulates a plethora of cellular proteins. In this study, we use tandem affinity purification and MS to identify host factors that interact with 11 different papillomavirus E2 proteins from diverse phylogenetic groups. The E2 proteins function in viral transcription and replication and correspondingly interact with host proteins involved in transcription, chromatin remodeling and modification, replication, and RNA processing. PMID:25758368

  12. An Interactive, Interdisciplinary, On-Line Graphics System for Presenting and Manipulating Directed Graphs.

    ERIC Educational Resources Information Center

    Beazley, William; And Others

    An interactive graphics system has been implemented for tutorial purposes and for research in man-machine communication of structural digraphs. An IMLAC intelligent terminal with ligthpen input is used in conjunction with a NOVA minicomputer. Successful application in linguistics and engineering problem solving are discussed, the latter in detail.…

  13. Globular and disordered—the non-identical twins in protein-protein interactions

    PubMed Central

    Teilum, Kaare; Olsen, Johan G.; Kragelund, Birthe B.

    2015-01-01

    In biology proteins from different structural classes interact across and within classes in ways that are optimized to achieve balanced functional outputs. The interactions between intrinsically disordered proteins (IDPs) and other proteins rely on changes in flexibility and this is seen as a strong determinant for their function. This has fostered the notion that IDP's bind with low affinity but high specificity. Here we have analyzed available detailed thermodynamic data for protein-protein interactions to put to the test if the thermodynamic profiles of IDP interactions differ from those of other protein-protein interactions. We find that ordered proteins and the disordered ones act as non-identical twins operating by similar principles but where the disordered proteins complexes are on average less stable by 2.5 kcal mol−1. PMID:26217672

  14. Globular and disordered-the non-identical twins in protein-protein interactions.

    PubMed

    Teilum, Kaare; Olsen, Johan G; Kragelund, Birthe B

    2015-01-01

    In biology proteins from different structural classes interact across and within classes in ways that are optimized to achieve balanced functional outputs. The interactions between intrinsically disordered proteins (IDPs) and other proteins rely on changes in flexibility and this is seen as a strong determinant for their function. This has fostered the notion that IDP's bind with low affinity but high specificity. Here we have analyzed available detailed thermodynamic data for protein-protein interactions to put to the test if the thermodynamic profiles of IDP interactions differ from those of other protein-protein interactions. We find that ordered proteins and the disordered ones act as non-identical twins operating by similar principles but where the disordered proteins complexes are on average less stable by 2.5 kcal mol(-1). PMID:26217672

  15. The protein interaction landscape of the human CMGC kinase group.

    PubMed

    Varjosalo, Markku; Keskitalo, Salla; Van Drogen, Audrey; Nurkkala, Helka; Vichalkovski, Anton; Aebersold, Ruedi; Gstaiger, Matthias

    2013-04-25

    Cellular information processing via reversible protein phosphorylation requires tight control of the localization, activity, and substrate specificity of protein kinases, which to a large extent is accomplished by complex formation with other proteins. Despite their critical role in cellular regulation and pathogenesis, protein interaction information is available for only a subset of the 518 human protein kinases. Here we present a global proteomic analysis of complexes of the human CMGC kinase group. In addition to subgroup-specific functional enrichment and modularity, the identified 652 high-confidence kinase-protein interactions provide a specific biochemical context for many poorly studied CMGC kinases. Furthermore, the analysis revealed a kinase-kinase subnetwork and candidate substrates for CMGC kinases. Finally, the presented interaction proteome uncovered a large set of interactions with proteins genetically linked to a range of human diseases, including cancer, suggesting additional routes for analyzing the role of CMGC kinases in controlling human disease pathways. PMID:23602568

  16. Membrane-Mediated Interaction between Strongly Anisotropic Protein Scaffolds

    PubMed Central

    Schweitzer, Yonatan; Kozlov, Michael M.

    2015-01-01

    Specialized proteins serve as scaffolds sculpting strongly curved membranes of intracellular organelles. Effective membrane shaping requires segregation of these proteins into domains and is, therefore, critically dependent on the protein-protein interaction. Interactions mediated by membrane elastic deformations have been extensively analyzed within approximations of large inter-protein distances, small extents of the protein-mediated membrane bending and small deviations of the protein shapes from isotropic spherical segments. At the same time, important classes of the realistic membrane-shaping proteins have strongly elongated shapes with large and highly anisotropic curvature. Here we investigated, computationally, the membrane mediated interaction between proteins or protein oligomers representing membrane scaffolds with strongly anisotropic curvature, and addressed, quantitatively, a specific case of the scaffold geometrical parameters characterizing BAR domains, which are crucial for membrane shaping in endocytosis. In addition to the previously analyzed contributions to the interaction, we considered a repulsive force stemming from the entropy of the scaffold orientation. We computed this interaction to be of the same order of magnitude as the well-known attractive force related to the entropy of membrane undulations. We demonstrated the scaffold shape anisotropy to cause a mutual aligning of the scaffolds and to generate a strong attractive interaction bringing the scaffolds close to each other to equilibrium distances much smaller than the scaffold size. We computed the energy of interaction between scaffolds of a realistic geometry to constitute tens of kBT, which guarantees a robust segregation of the scaffolds into domains. PMID:25710602

  17. 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

  18. Synthetic protein interactions reveal a functional map of the cell

    PubMed Central

    Berry, Lisa K; Ólafsson, Guðjón; Ledesma-Fernández, Elena; Thorpe, Peter H

    2016-01-01

    To understand the function of eukaryotic cells, it is critical to understand the role of protein-protein interactions and protein localization. Currently, we do not know the importance of global protein localization nor do we understand to what extent the cell is permissive for new protein associations – a key requirement for the evolution of new protein functions. To answer this question, we fused every protein in the yeast Saccharomyces cerevisiae with a partner from each of the major cellular compartments and quantitatively assessed the effects upon growth. This analysis reveals that cells have a remarkable and unanticipated tolerance for forced protein associations, even if these associations lead to a proportion of the protein moving compartments within the cell. Furthermore, the interactions that do perturb growth provide a functional map of spatial protein regulation, identifying key regulatory complexes for the normal homeostasis of eukaryotic cells. DOI: http://dx.doi.org/10.7554/eLife.13053.001 PMID:27098839

  19. Protein Interactions in Genome Maintenance as Novel Antibacterial Targets

    PubMed Central

    Walsh, Brian W.; Shapiro, Walker; Simmons, Lyle A.; Keck, James L.

    2013-01-01

    Antibacterial compounds typically act by directly inhibiting essential bacterial enzyme activities. Although this general mechanism of action has fueled traditional antibiotic discovery efforts for decades, new antibiotic development has not kept pace with the emergence of drug resistant bacterial strains. These limitations have severely restricted the therapeutic tools available for treating bacterial infections. Here we test an alternative antibacterial lead-compound identification strategy in which essential protein-protein interactions are targeted rather than enzymatic activities. Bacterial single-stranded DNA-binding proteins (SSBs) form conserved protein interaction “hubs” that are essential for recruiting many DNA replication, recombination, and repair proteins to SSB/DNA nucleoprotein substrates. Three small molecules that block SSB/protein interactions are shown to have antibacterial activity against diverse bacterial species. Consistent with a model in which the compounds target multiple SSB/protein interactions, treatment of Bacillus subtilis cultures with the compounds leads to rapid inhibition of DNA replication and recombination, and ultimately to cell death. The compounds also have unanticipated effects on protein synthesis that could be due to a previously unknown role for SSB/protein interactions in translation or to off-target effects. Our results highlight the potential of targeting protein-protein interactions, particularly those that mediate genome maintenance, as a powerful approach for identifying new antibacterial compounds. PMID:23536821

  20. Genome-wide protein-protein interactions and protein function exploration in cyanobacteria.

    PubMed

    Lv, Qi; Ma, Weimin; Liu, Hui; Li, Jiang; Wang, Huan; Lu, Fang; Zhao, Chen; Shi, Tieliu

    2015-01-01

    Genome-wide network analysis is well implemented to study proteins of unknown function. Here, we effectively explored protein functions and the biological mechanism based on inferred high confident protein-protein interaction (PPI) network in cyanobacteria. We integrated data from seven different sources and predicted 1,997 PPIs, which were evaluated by experiments in molecular mechanism, text mining of literatures in proved direct/indirect evidences, and "interologs" in conservation. Combined the predicted PPIs with known PPIs, we obtained 4,715 no-redundant PPIs (involving 3,231 proteins covering over 90% of genome) to generate the PPI network. Based on the PPI network, terms in Gene ontology (GO) were assigned to function-unknown proteins. Functional modules were identified by dissecting the PPI network into sub-networks and analyzing pathway enrichment, with which we investigated novel function of underlying proteins in protein complexes and pathways. Examples of photosynthesis and DNA repair indicate that the network approach is a powerful tool in protein function analysis. Overall, this systems biology approach provides a new insight into posterior functional analysis of PPIs in cyanobacteria. PMID:26490033

  1. Genome-wide protein-protein interactions and protein function exploration in cyanobacteria

    PubMed Central

    Lv, Qi; Ma, Weimin; Liu, Hui; Li, Jiang; Wang, Huan; Lu, Fang; Zhao, Chen; Shi, Tieliu

    2015-01-01

    Genome-wide network analysis is well implemented to study proteins of unknown function. Here, we effectively explored protein functions and the biological mechanism based on inferred high confident protein-protein interaction (PPI) network in cyanobacteria. We integrated data from seven different sources and predicted 1,997 PPIs, which were evaluated by experiments in molecular mechanism, text mining of literatures in proved direct/indirect evidences, and “interologs” in conservation. Combined the predicted PPIs with known PPIs, we obtained 4,715 no-redundant PPIs (involving 3,231 proteins covering over 90% of genome) to generate the PPI network. Based on the PPI network, terms in Gene ontology (GO) were assigned to function-unknown proteins. Functional modules were identified by dissecting the PPI network into sub-networks and analyzing pathway enrichment, with which we investigated novel function of underlying proteins in protein complexes and pathways. Examples of photosynthesis and DNA repair indicate that the network approach is a powerful tool in protein function analysis. Overall, this systems biology approach provides a new insight into posterior functional analysis of PPIs in cyanobacteria. PMID:26490033

  2. All-optical manipulation and probing of the d–f exchange interaction in EuTe

    PubMed Central

    Subkhangulov, R. R.; Henriques, A. B.; Rappl, P. H. O.; Abramof, E.; Rasing, Th.; Kimel, A. V.

    2014-01-01

    We demonstrate that the ultrafast fast dynamics of the d–f exchange interaction, between conduction band electrons and lattice spins in EuTe, can be accessed using an all-optical technique. Our results reveal, in full detail, the time evolution of the d–f exchange interaction induced by a femtosecond laser pulse. Specifically, by monitoring the time resolved dynamics of the reflectivity changes and Kerr rotation of a weak light pulse reflected from the surface of the sample, it is shown that an intense femtosecond light pulse with photon energies higher than that of the bandgap, triggers spin waves in EuTe. The laser-induced spin waves modulate the d–f exchange interaction, and cause the bandgap to oscillate with an amplitude reaching 1 meV, at frequencies up to tens of GHz. The ability to control and monitor the dynamics of the exchange energy with our all-optical technique opens up new opportunities for the manipulation of magnetism at ultrafast time-scales. PMID:24658449

  3. All-optical manipulation and probing of the d-f exchange interaction in EuTe.

    PubMed

    Subkhangulov, R R; Henriques, A B; Rappl, P H O; Abramof, E; Rasing, Th; Kimel, A V

    2014-01-01

    We demonstrate that the ultrafast fast dynamics of the d-f exchange interaction, between conduction band electrons and lattice spins in EuTe, can be accessed using an all-optical technique. Our results reveal, in full detail, the time evolution of the d-f exchange interaction induced by a femtosecond laser pulse. Specifically, by monitoring the time resolved dynamics of the reflectivity changes and Kerr rotation of a weak light pulse reflected from the surface of the sample, it is shown that an intense femtosecond light pulse with photon energies higher than that of the bandgap, triggers spin waves in EuTe. The laser-induced spin waves modulate the d-f exchange interaction, and cause the bandgap to oscillate with an amplitude reaching 1 meV, at frequencies up to tens of GHz. The ability to control and monitor the dynamics of the exchange energy with our all-optical technique opens up new opportunities for the manipulation of magnetism at ultrafast time-scales. PMID:24658449

  4. Design of Protein-Protein Interactions with a Novel Ensemble-Based Scoring Algorithm

    NASA Astrophysics Data System (ADS)

    Roberts, Kyle E.; Cushing, Patrick R.; Boisguerin, Prisca; Madden, Dean R.; Donald, Bruce R.

    Protein-protein interactions (PPIs) are vital for cell signaling, protein trafficking and localization, gene expression, and many other biological functions. Rational modification of PPI targets provides a mechanism to understand their function and importance. However, PPI systems often have many more degrees of freedom and flexibility than the small-molecule binding sites typically targeted by protein design algorithms. To handle these challenging design systems, we have built upon the computational protein design algorithm K * [8,19] to develop a new design algorithm to study protein-protein and protein-peptide interactions. We validated our algorithm through the design and experimental testing of novel peptide inhibitors.

  5. CHARMM-GUI PDB Manipulator for Advanced Modeling and Simulations of Proteins Containing Non-standard Residues

    PubMed Central

    Jo, Sunhwan; Cheng, Xi; Islam, Shahidul M.; Huang, Lei; Rui, Huan; Zhu, Allen; Lee, Hui Sun; Qi, Yifei; Han, Wei; Vanommeslaeghe, Kenno; MacKerell, Alexander D.; Roux, Benoît; Im, Wonpil

    2016-01-01

    CHARMM-GUI, http://www.charmm-gui.org, is a web-based graphical user interface to prepare molecular simulation systems and input files to facilitate the usage of common and advanced simulation techniques. Since its original development in 2006, CHARMM-GUI has been widely adopted for various purposes and now contains a number of different modules designed to setup a broad range of simulations including free energy calculation and large-scale coarse-grained representation. Here, we describe functionalities that have recently been integrated into CHARMM-GUI PDB Manipulator, such as ligand force field generation, incorporation of methanethiosulfonate (MTS) spin labels and chemical modifiers, and substitution of amino acids with unnatural amino acids. These new features are expected to be useful in advanced biomolecular modeling and simulation of proteins. PMID:25443960

  6. Environmental manipulations early in development alter seizure activity, Ih and HCN1 protein expression later in life.

    PubMed

    Schridde, Ulrich; Strauss, Ulf; Bräuer, Anja U; van Luijtelaar, Gilles

    2006-06-01

    Although absence epilepsy has a genetic origin, evidence from an animal model (Wistar Albino Glaxo/Rijswijk; WAG/Rij) suggests that seizures are sensitive to environmental manipulations. Here, we show that manipulations of the early rearing environment (neonatal handling, maternal deprivation) of WAG/Rij rats leads to a pronounced decrease in seizure activity later in life. Recent observations link seizure activity in WAG/Rij rats to the hyperpolarization-activated cation current (Ih) in the somatosensory cortex, the site of seizure generation. Therefore, we investigated whether the alterations in seizure activity between rats reared differently might be correlated with changes in Ih and its channel subunits hyperpolarization-activated cation channel HCN1, 2 and 4. Whole-cell recordings from layer 5 pyramidal neurons, in situ hybridization and Western blot of the somatosensory cortex revealed an increase in Ih and HCN1 in neonatal handled and maternal deprived, compared to control rats. The increase was specific to HCN1 protein expression and did not involve HCN2/4 protein expression, or mRNA expression of any of the subunits (HCN1, 2, 4). Our findings provide the first evidence that relatively mild changes in the neonatal environment have a long-term impact of absence seizures, Ih and HCN1, and suggest that an increase of Ih and HCN1 is associated with absence seizure reduction. Our findings shed new light on the role of Ih and HCN in brain functioning and development and demonstrate that genetically determined absence seizures are quite sensitive for early interventions. PMID:16820024

  7. Visualization and targeted disruption of protein interactions in living cells.

    PubMed

    Herce, Henry D; Deng, Wen; Helma, Jonas; Leonhardt, Heinrich; Cardoso, M Cristina

    2013-01-01

    Protein-protein interactions are the basis of all processes in living cells, but most studies of these interactions rely on biochemical in vitro assays. Here we present a simple and versatile fluorescent-three-hybrid (F3H) strategy to visualize and target protein-protein interactions. A high-affinity nanobody anchors a GFP-fusion protein of interest at a defined cellular structure and the enrichment of red-labelled interacting proteins is measured at these sites. With this approach, we visualize the p53-HDM2 interaction in living cells and directly monitor the disruption of this interaction by Nutlin 3, a drug developed to boost p53 activity in cancer therapy. We further use this approach to develop a cell-permeable vector that releases a highly specific peptide disrupting the p53 and HDM2 interaction. The availability of multiple anchor sites and the simple optical readout of this nanobody-based capture assay enable systematic and versatile analyses of protein-protein interactions in practically any cell type and species. PMID:24154492

  8. GWIDD: a comprehensive resource for genome-wide structural modeling of protein-protein interactions

    PubMed Central

    2012-01-01

    Protein-protein interactions are a key component of life processes. The knowledge of the three-dimensional structure of these interactions is important for understanding protein function. Genome-Wide Docking Database (http://gwidd.bioinformatics.ku.edu) offers an extensive source of data for structural studies of protein-protein complexes on genome scale. The current release of the database combines the available experimental data on the structure and characteristics of protein interactions with structural modeling of protein complexes for 771 organisms spanned over the entire universe of life from viruses to humans. The interactions are stored in a relational database with user-friendly interface that includes various search options. The search results can be interactively previewed; the structures, downloaded, along with the interaction characteristics. PMID:23245398

  9. Experimental manipulation of compaction of the mouse embryo alters patterns of protein phosphorylation

    SciTech Connect

    Bloom, T. )

    1991-03-01

    Compaction, occurring at the eight-cell stage of mouse development, is the process of cell flattening and polarisation by which cellular asymmetry is first established. Changes in the pattern of protein phosphorylation have been correlated with this early event of development. In the study reported here, groups of embryos were treated in ways known to affect particular features of compaction and were then labeled with ({sup 32}P)orthophosphate; the phosphoproteins obtained were examined following electrophoresis in one and two dimensions. Four-cell embryos were treated with protein synthesis inhibitors, which advance cell flattening. This treatment resulted in only minor differences from the phosphoprotein profile of untreated four-cell embryos. Inhibition of protein synthesis at the eight-cell stage has little effect on cell flattening or polarisation. However, some phosphoproteins that are observed normally in eight-cell but not in four-cell embryos were no longer detectable if labeling took place in the presence of protein synthesis inhibitors. Eight-cell embryos incubated in phorbol 12-myristate 13-acetate, which disrupts various features of compaction, showed a relative increase in the phosphorylation of a group of phosphoprotein spots associated with the eight-cell but not with the four-cell stage. Embryos incubated in Ca2(+)-free medium, which prevents intercellular flattening and delays polarisation, showed a relative decrease in the phosphorylation of the same group of phosphoprotein spots. The behaviour of these phosphoproteins may therefore be correlated with some of the features of compaction.

  10. Using Simple Manipulatives to Improve Student Comprehension of a Complex Biological Process: Protein Synthesis

    ERIC Educational Resources Information Center

    Guzman, Karen; Bartlett, John

    2012-01-01

    Biological systems and living processes involve a complex interplay of biochemicals and macromolecular structures that can be challenging for undergraduate students to comprehend and, thus, misconceptions abound. Protein synthesis, or translation, is an example of a biological process for which students often hold many misconceptions. This article…

  11. PIMA: Protein-Protein interactions in Macromolecular Assembly - a web server for its Analysis and Visualization

    PubMed Central

    Kaleeckal Mathew, Oommen; Sowdhamini, Ramanathan

    2016-01-01

    Protein-protein interactions are essential for the basic biological machinery of the cell. This is important for processes like protein synthesis, enzyme kinetics, molecular assembly and signal transduction. A high number of macromolecular structural complexes are known due to recent advances in structure determination techniques. Therefore, it is of interest to develop an interactive tool to objectively analyze large protein complexes. Hence, we describe the development and utility of a web enabled application named ‘Protein-Protein Interaction in Macro-molecular Assembly’ (PIMA) for the analysis of large protein assemblies. The intricate details of physical interactions amongst protein subunits in a large complex are presented as simple user preferred interactive network diagrams PMID:27212837

  12. Analyzing Protein-Phosphoinositide Interactions with Liposome Flotation Assays.

    PubMed

    Busse, Ricarda A; Scacioc, Andreea; Schalk, Amanda M; Krick, Roswitha; Thumm, Michael; Kühnel, Karin

    2016-01-01

    Liposome flotation assays are a convenient tool to study protein-phosphoinositide interactions. Working with liposomes resembles physiological conditions more than protein-lipid overlay assays, which makes this method less prone to detect false positive interactions. However, liposome lipid composition must be well-considered in order to prevent nonspecific binding of the protein through electrostatic interactions with negatively charged lipids like phosphatidylserine. In this protocol we use the PROPPIN Hsv2 (homologous with swollen vacuole phenotype 2) as an example to demonstrate the influence of liposome lipid composition on binding and show how phosphoinositide binding specificities of a protein can be characterized with this method. PMID:26552682

  13. A Novel Secreted Protein, MYR1, Is Central to Toxoplasma’s Manipulation of Host Cells

    PubMed Central

    Franco, Magdalena; Panas, Michael W.; Marino, Nicole D.; Lee, Mei-Chong Wendy; Buchholz, Kerry R.; Kelly, Felice D.; Bednarski, Jeffrey J.; Sleckman, Barry P.; Pourmand, Nader

    2016-01-01

    ABSTRACT The intracellular protozoan Toxoplasma gondii dramatically reprograms the transcriptome of host cells it infects, including substantially up-regulating the host oncogene c-myc. By applying a flow cytometry-based selection to infected mouse cells expressing green fluorescent protein fused to c-Myc (c-Myc–GFP), we isolated mutant tachyzoites defective in this host c-Myc up-regulation. Whole-genome sequencing of three such mutants led to the identification of MYR1 (Myc regulation 1; TGGT1_254470) as essential for c-Myc induction. MYR1 is a secreted protein that requires TgASP5 to be cleaved into two stable portions, both of which are ultimately found within the parasitophorous vacuole and at the parasitophorous vacuole membrane. Deletion of MYR1 revealed that in addition to its requirement for c-Myc up-regulation, the MYR1 protein is needed for the ability of Toxoplasma tachyzoites to modulate several other important host pathways, including those mediated by the dense granule effectors GRA16 and GRA24. This result, combined with its location at the parasitophorous vacuole membrane, suggested that MYR1 might be a component of the machinery that translocates Toxoplasma effectors from the parasitophorous vacuole into the host cytosol. Support for this possibility was obtained by showing that transit of GRA24 to the host nucleus is indeed MYR1-dependent. As predicted by this pleiotropic phenotype, parasites deficient in MYR1 were found to be severely attenuated in a mouse model of infection. We conclude, therefore, that MYR1 is a novel protein that plays a critical role in how Toxoplasma delivers effector proteins to the infected host cell and that this is crucial to virulence. PMID:26838724

  14. Mitochondrial nucleoid interacting proteins support mitochondrial protein synthesis

    PubMed Central

    He, J.; Cooper, H. M.; Reyes, A.; Di Re, M.; Sembongi, H.; Litwin, T. R.; Gao, J.; Neuman, K. C.; Fearnley, I. M.; Spinazzola, A.; Walker, J. E.; Holt, I. J.

    2012-01-01

    Mitochondrial ribosomes and translation factors co-purify with mitochondrial nucleoids of human cells, based on affinity protein purification of tagged mitochondrial DNA binding proteins. Among the most frequently identified proteins were ATAD3 and prohibitin, which have been identified previously as nucleoid components, using a variety of methods. Both proteins are demonstrated to be required for mitochondrial protein synthesis in human cultured cells, and the major binding partner of ATAD3 is the mitochondrial ribosome. Altered ATAD3 expression also perturbs mtDNA maintenance and replication. These findings suggest an intimate association between nucleoids and the machinery of protein synthesis in mitochondria. ATAD3 and prohibitin are tightly associated with the mitochondrial membranes and so we propose that they support nucleic acid complexes at the inner membrane of the mitochondrion. PMID:22453275

  15. Widespread Expansion of Protein Interaction Capabilities by Alternative Splicing.

    PubMed

    Yang, Xinping; Coulombe-Huntington, Jasmin; Kang, Shuli; Sheynkman, Gloria M; Hao, Tong; Richardson, Aaron; Sun, Song; Yang, Fan; Shen, Yun A; Murray, Ryan R; Spirohn, Kerstin; Begg, Bridget E; Duran-Frigola, Miquel; MacWilliams, Andrew; Pevzner, Samuel J; Zhong, Quan; Trigg, Shelly A; Tam, Stanley; Ghamsari, Lila; Sahni, Nidhi; Yi, Song; Rodriguez, Maria D; Balcha, Dawit; Tan, Guihong; Costanzo, Michael; Andrews, Brenda; Boone, Charles; Zhou, Xianghong J; Salehi-Ashtiani, Kourosh; Charloteaux, Benoit; Chen, Alyce A; Calderwood, Michael A; Aloy, Patrick; Roth, Frederick P; Hill, David E; Iakoucheva, Lilia M; Xia, Yu; Vidal, Marc

    2016-02-11

    While alternative splicing is known to diversify the functional characteristics of some genes, the extent to which protein isoforms globally contribute to functional complexity on a proteomic scale remains unknown. To address this systematically, we cloned full-length open reading frames of alternatively spliced transcripts for a large number of human genes and used protein-protein interaction profiling to functionally compare hundreds of protein isoform pairs. The majority of isoform pairs share less than 50% of their interactions. In the global context of interactome network maps, alternative isoforms tend to behave like distinct proteins rather than minor variants of each other. Interaction partners specific to alternative isoforms tend to be expressed in a highly tissue-specific manner and belong to distinct functional modules. Our strategy, applicable to other functional characteristics, reveals a widespread expansion of protein interaction capabilities through alternative splicing and suggests that many alternative "isoforms" are functionally divergent (i.e., "functional alloforms"). PMID:26871637

  16. Computational biology for target discovery and characterization: a feasibility study in protein-protein interaction detection

    SciTech Connect

    Zhou, C; Zemla, A

    2009-02-25

    In this work we developed new code for detecting putative multi-domain protein-protein interactions for a small network of bacterial pathogen proteins, and determined how structure-driven domain-fusion (DF) methods should be scaled up for whole-proteome analysis. Protein-protein interactions are of great interest in structural biology and are important for understanding the biology of pathogens. The ability to predict protein-protein interactions provides a means for development of anti-microbials that may interfer with key processes in pathogenicity. The function of a protein-protein complex can be elucidated through knowledge of its structure. The overall goal of this project was to determine the feasibility of extending current LLNL capabilities to produce a high-throughput systems bio-informatics capability for identification and characterization of putative interacting protein partners within known or suspected small protein networks. We extended an existing LLNL methodology for identification of putative protein-protein interacting partners (Chakicherla et al (in review)) by writing a new code to identify multi-domain-fusion linkages (3 or more per complex). We applied these codes to the proteins in the Yersinia pestis quorum sensing network, known as the lsr operon, which comprises a virulence mechanism in this pathogen. We determined that efficient application of our computational algorithms in high-throughput for detection of putative protein-protein complexes genome wide would require pre-computation of PDB domains and construction of a domain-domain association database.

  17. Structural study of surfactant-dependent interaction with protein

    SciTech Connect

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

    2015-06-24

    Small-angle neutron scattering (SANS) has been used to study the complex structure of anionic BSA protein with three different (cationic DTAB, anionic SDS and non-ionic C12E10) surfactants. These systems form very different surfactant-dependent complexes. We show that the structure of protein-surfactant complex is initiated by the site-specific electrostatic interaction between the components, followed by the hydrophobic interaction at high surfactant concentrations. It is also found that hydrophobic interaction is preferred over the electrostatic interaction in deciding the resultant structure of protein-surfactant complexes.

  18. On Manipulating Nonverbal Interaction Style to Increase Anthropomorphic Computer Character Credibility

    SciTech Connect

    Cowell, Andrew J.; Stanney, Kay M.

    2003-09-01

    This study examined the effectiveness of enhancing humanagentinteraction through the use of nonverbal behaviors. Ataxonomy is described, which organizes nonverbal behaviorsinto functional categories and the manner in which they can beembodied (i.e. through gesture, posture, paralanguage, eyecontact and facial expression). Prototype computer characterswere created according to guidelines extracted from thetaxonomy and their efficacy was empirical evaluated. Theresults indicate that by including trusting nonverbal behaviors,the perceived credibility of a computer character was enhanced,although addition of trusting bodily nonverbal behaviorprovided little in addition to trusting facial nonverbal behavior.Perhaps more importantly, a character expressing non-trustingnonverbal behaviors was perceived to be the least credible of allcharacters examined (including a character that expressed nononverbal behavior). Participants that interacted with thispersona perceived the task to be more demanding, madesignificantly more errors, and rated their interaction lesspositively and more monotonous than those using trustingpersonas. They also rated this character to be less likable,accurate, and intelligent. Taken together, the results from thisstudy suggest that there may indeed be benefit to endowingcomputer characters with nonverbal trusting behaviors, as longas those behaviors are accurately and appropriately portrayed.Such behaviors may lead to a more trusting environment andpositive experience for users. Negative character behavior,however, such as non-trusting behavior, may squander theadvantages that embodiment brings.

  19. Manipulating Magnetism: Ru-2(5+) Paddlewheels Devoid of Axial Interactions

    SciTech Connect

    Chiarella, Gina M; Cotton, F. A.; Murillo, Carlos A; Ventura, Karen; Vilagran, Dino; Wang, Xiaoping

    2014-01-01

    Variable-temperature magnetic and structural data of two pairs of diruthenium isomers, one pair having an axial ligand and the formula Ru-2(DArF)(4)Cl (where DArF is the anion of a diarylformamidine isomer and Ar = p-anisyl or m-anisyl) and the other one being essentially identical but devoid of axial ligands and having the formula [Ru-2(DArF)(4)]BF4, show that the axial ligand has a significant effect on the electronic structure of the diruthenium unit. Variable temperature crystallographic and magnetic data as well as density functional theory calculations unequivocally demonstrate the occurrence of pi interactions between the p orbitals of the chlorine ligand and the pi* orbitals in the Ru-2(5+) units. The magnetic and structural data are consistent with the existence of combined ligand sigma/metal sigma and ligand p pi/metal-d pi interactions. Electron paramagnetic resonance data show unambiguously that the unpaired electrons are in metal-based molecular orbitals.

  20. PPLook: an automated data mining tool for protein-protein interaction

    PubMed Central

    2010-01-01

    Background Extracting and visualizing of protein-protein interaction (PPI) from text literatures are a meaningful topic in protein science. It assists the identification of interactions among proteins. There is a lack of tools to extract PPI, visualize and classify the results. Results We developed a PPI search system, termed PPLook, which automatically extracts and visualizes protein-protein interaction (PPI) from text. Given a query protein name, PPLook can search a dataset for other proteins interacting with it by using a keywords dictionary pattern-matching algorithm, and display the topological parameters, such as the number of nodes, edges, and connected components. The visualization component of PPLook enables us to view the interaction relationship among the proteins in a three-dimensional space based on the OpenGL graphics interface technology. PPLook can also provide the functions of selecting protein semantic class, counting the number of semantic class proteins which interact with query protein, counting the literature number of articles appearing the interaction relationship about the query protein. Moreover, PPLook provides heterogeneous search and a user-friendly graphical interface. Conclusions PPLook is an effective tool for biologists and biosystem developers who need to access PPI information from the literature. PPLook is freely available for non-commercial users at http://meta.usc.edu/softs/PPLook. PMID:20550717

  1. An ontology-based search engine for protein-protein interactions

    PubMed Central

    2010-01-01

    Background Keyword matching or ID matching is the most common searching method in a large database of protein-protein interactions. They are purely syntactic methods, and retrieve the records in the database that contain a keyword or ID specified in a query. Such syntactic search methods often retrieve too few search results or no results despite many potential matches present in the database. Results We have developed a new method for representing protein-protein interactions and the Gene Ontology (GO) using modified Gödel numbers. This representation is hidden from users but enables a search engine using the representation to efficiently search protein-protein interactions in a biologically meaningful way. Given a query protein with optional search conditions expressed in one or more GO terms, the search engine finds all the interaction partners of the query protein by unique prime factorization of the modified Gödel numbers representing the query protein and the search conditions. Conclusion Representing the biological relations of proteins and their GO annotations by modified Gödel numbers makes a search engine efficiently find all protein-protein interactions by prime factorization of the numbers. Keyword matching or ID matching search methods often miss the interactions involving a protein that has no explicit annotations matching the search condition, but our search engine retrieves such interactions as well if they satisfy the search condition with a more specific term in the ontology. PMID:20122195

  2. 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

  3. Interaction between Vaccinium bracteatum Thunb. leaf pigment and rice proteins.

    PubMed

    Wang, Li; Xu, Yuan; Zhou, Sumei; Qian, Haifeng; Zhang, Hui; Qi, Xiguang; Fan, Meihua

    2016-03-01

    In this study, we investigated the interaction of Vaccinium bracteatum Thunb. leaf (VBTL) pigment and rice proteins. In the presence of rice protein, VBTL pigment antioxidant activity and free polyphenol content decreased by 67.19% and 68.11%, respectively, and L(∗) of the protein-pigment complex decreased significantly over time. L(∗) values of albumin, globulin and glutelin during 60-min pigment exposure decreased by 55.00, 57.14, and 54.30%, respectively, indicating that these proteins had bound to the pigment. A significant difference in protein surface hydrophobicity was observed between rice proteins and pigment-protein complexes, indicating that hydrophobic interaction is a major binding mechanism between VBTL pigment and rice proteins. A significant difference in secondary structures between proteins and protein-pigment complexes was also uncovered, indicating that hydrogen bonding may be another mode of interaction between VBTL pigment and rice proteins. Our results indicate that VBTL pigment can stain rice proteins with hydrophobic and hydrogen interactions. PMID:26471554

  4. Making the LINC: SUN and KASH protein interactions

    PubMed Central

    Kim, Dae In; Birendra, KC; Roux, Kyle J.

    2015-01-01

    Cell nuclei are physically integrated with the cytoskeleton through the LINC complex (for LInker of Nucleoskeleton and Cytoskeleton), a structure that spans the nuclear envelope to link the nucleoskeleton and cytoskeleton. Outer nuclear membrane KASH domain proteins and inner nuclear membrane SUN domain proteins interact to form the core of the LINC complex. In this review we provide a comprehensive analysis of the reported protein-protein interactions for KASH and SUN domain proteins. This critical structure, directly connecting the genome with the rest of the cell, contributes to a myriad of cellular functions and, when perturbed, is associated with human disease. PMID:25720065

  5. Interacting proteins as genetic modifiers of Huntington disease.

    PubMed

    Li, Xiao-Jiang; Friedman, Meyer; Li, Shihua

    2007-11-01

    Huntington disease is caused by polyglutamine expansion in huntingtin, a 350 kD protein that is ubiquitously expressed and widely distributed at the subcellular level. Recently, Kaltenbach et al. identified a large collection of novel huntingtin-interacting proteins, several of which modify mutant huntingtin toxicity in Drosophila. Thus, the interaction of mutant huntingtin with certain protein partners can influence its toxicity and therefore the severity and/or progression of Huntington disease. PMID:17961788

  6. Protein lethality investigated in terms of long range dynamical interactions.

    PubMed

    Rodrigues, Francisco A; Costa, Luciano da Fontoura

    2009-04-01

    The relationship between network structure/dynamics and biological function constitutes a fundamental issue in systems biology. However, despite many related investigations, the correspondence between structure and biological functions is not yet fully understood. A related subject that has deserved particular attention recently concerns how essentiality is related to the structure and dynamics of protein interactions. In the current work, protein essentiality is investigated in terms of long range influences in protein-protein interaction networks by considering simulated dynamical aspects. This analysis is performed with respect to outward activations, an approach which models the propagation of interactions between proteins by considering self-avoiding random walks. The obtained results are compared to protein local connectivity. Both the connectivity and the outward activations were found to be strongly related to protein essentiality. PMID:19396375

  7. Rosetta stone method for detecting protein function and protein-protein interactions from genome sequences

    DOEpatents

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

    2002-10-15

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

  8. Structural Similarity and Classification of Protein Interaction Interfaces

    PubMed Central

    Zhao, Nan; Pang, Bin; Shyu, Chi-Ren; Korkin, Dmitry

    2011-01-01

    Interactions between proteins play a key role in many cellular processes. Studying protein-protein interactions that share similar interaction interfaces may shed light on their evolution and could be helpful in elucidating the mechanisms behind stability and dynamics of the protein complexes. When two complexes share structurally similar subunits, the similarity of the interaction interfaces can be found through a structural superposition of the subunits. However, an accurate detection of similarity between the protein complexes containing subunits of unrelated structure remains an open problem. Here, we present an alignment-free machine learning approach to measure interface similarity. The approach relies on the feature-based representation of protein interfaces and does not depend on the superposition of the interacting subunit pairs. Specifically, we develop an SVM classifier of similar and dissimilar interfaces and derive a feature-based interface similarity measure. Next, the similarity measure is applied to a set of 2,806×2,806 binary complex pairs to build a hierarchical classification of protein-protein interactions. Finally, we explore case studies of similar interfaces from each level of the hierarchy, considering cases when the subunits forming interactions are either homologous or structurally unrelated. The analysis has suggested that the positions of charged residues in the homologous interfaces are not necessarily conserved and may exhibit more complex conservation patterns. PMID:21589874

  9. Molecular interactions of graphene oxide with human blood plasma proteins

    NASA Astrophysics Data System (ADS)

    Kenry, Affa Affb Affc; Loh, Kian Ping; Lim, Chwee Teck

    2016-04-01

    We investigate the molecular interactions between graphene oxide (GO) and human blood plasma proteins. To gain an insight into the bio-physico-chemical activity of GO in biological and biomedical applications, we performed a series of biophysical assays to quantify the molecular interactions between GO with different lateral size distributions and the three essential human blood plasma proteins. We elucidate the various aspects of the GO-protein interactions, particularly, the adsorption, binding kinetics and equilibrium, and conformational stability, through determination of quantitative parameters, such as GO-protein association constants, binding cooperativity, and the binding-driven protein structural changes. We demonstrate that the molecular interactions between GO and plasma proteins are significantly dependent on the lateral size distribution and mean lateral sizes of the GO nanosheets and their subtle variations may markedly influence the GO-protein interactions. Consequently, we propose the existence of size-dependent molecular interactions between GO nanosheets and plasma proteins, and importantly, the presence of specific critical mean lateral sizes of GO nanosheets in achieving very high association and fluorescence quenching efficiency of the plasma proteins. We anticipate that this work will provide a basis for the design of graphene-based and other related nanomaterials for a plethora of biological and biomedical applications.

  10. Proteins interacting with Membranes: Protein Sorting and Membrane Shaping

    NASA Astrophysics Data System (ADS)

    Callan-Jones, Andrew

    2015-03-01

    Membrane-bound transport in cells requires generating membrane curvature. In addition, transport is selective, in order to establish spatial gradients of membrane components in the cell. The mechanisms underlying cell membrane shaping by proteins and the influence of curvature on membrane composition are active areas of study in cell biophysics. In vitro approaches using Giant Unilamellar Vesicles (GUVs) are a useful tool to identify the physical mechanisms that drive sorting of membrane components and membrane shape change by proteins. I will present recent work on the curvature sensing and generation of IRSp53, a protein belonging to the BAR family, whose members, sharing a banana-shaped backbone, are involved in endocytosis. Pulling membrane tubes with 10-100 nm radii from GUVs containing encapsulated IRSp53 have, unexpectedly, revealed a non-monotonic dependence of the protein concentration on the tube as a function of curvature. Experiments also show that bound proteins alter the tube mechanics and that protein phase separation along the tube occurs at low tensions. I will present accompanying theoretical work that can explain these findings based on the competition between the protein's intrinsic curvature and the effective rigidity of a membrane-protein patch.

  11. The interaction of IQGAPs with calmodulin-like proteins.

    PubMed

    Pathmanathan, Sevvel; Hamilton, Elaine; Atcheson, Erwan; Timson, David J

    2011-04-01

    Since their identification over 15 years ago, the IQGAP (IQ-motif-containing GTPase-activating protein) family of proteins have been implicated in a wide range of cellular processes, including cytoskeletal reorganization, cell-cell adhesion, cytokinesis and apoptosis. These processes rely on protein-protein interactions, and understanding these (and how they influence one another) is critical in determining how the IQGAPs function. A key group of interactions is with calmodulin and the structurally related proteins myosin essential light chain and S100B. These interactions occur primarily through a series of IQ motifs, which are α-helical segments of the protein located towards the middle of the primary sequence. The three human IQGAP isoforms (IQGAP1, IQGAP2 and IQGAP3) all have four IQ motifs. However, these have different affinities for calmodulin, myosin light chain and S100B. Whereas all four IQ motifs of IQGAP1 interact with calmodulin in the presence of calcium, only the last two do so in the absence of calcium. IQ1 (the first IQ motif) interacts with the myosin essential light chain Mlc1sa and the first two undergo a calcium-dependent interaction with S100B. The significance of the interaction between Mlc1sa and IQGAP1 in mammals is unknown. However, a similar interaction involving the Saccharomyces cerevisiae IQGAP-like protein Iqg1p is involved in cytokinesis, leading to speculation that there may be a similar role in mammals. PMID:21428964

  12. Information-driven structural modelling of protein-protein interactions.

    PubMed

    Rodrigues, João P G L M; Karaca, Ezgi; Bonvin, Alexandre M J J

    2015-01-01

    Protein-protein docking aims at predicting the three-dimensional structure of a protein complex starting from the free forms of the individual partners. As assessed in the CAPRI community-wide experiment, the most successful docking algorithms combine pure laws of physics with information derived from various experimental or bioinformatics sources. Of these so-called "information-driven" approaches, HADDOCK stands out as one of the most successful representatives. In this chapter, we briefly summarize which experimental information can be used to drive the docking prediction in HADDOCK, and then focus on the docking protocol itself. We discuss and illustrate with a tutorial example a "classical" protein-protein docking prediction, as well as more recent developments for modelling multi-body systems and large conformational changes. PMID:25330973

  13. Actin Interacts with Dengue Virus 2 and 4 Envelope Proteins

    PubMed Central

    Jitoboam, Kunlakanya; Phaonakrop, Narumon; Libsittikul, Sirikwan; Thepparit, Chutima; Roytrakul, Sittiruk; Smith, Duncan R.

    2016-01-01

    Dengue virus (DENV) remains a significant public health problem in many tropical and sub-tropical countries worldwide. The DENV envelope (E) protein is the major antigenic determinant and the protein that mediates receptor binding and endosomal fusion. In contrast to some other DENV proteins, relatively few cellular interacting proteins have been identified. To address this issue a co-immuoprecipitation strategy was employed. The predominant co-immunoprecipitating proteins identified were actin and actin related proteins, however the results suggested that actin was the only bona fide interacting partner. Actin was shown to interact with the E protein of DENV 2 and 4, and the interaction between actin and DENV E protein was shown to occur in a truncated DENV consisting of only domains I and II. Actin was shown to decrease during infection, but this was not associated with a decrease in gene transcription. Actin-related proteins also showed a decrease in expression during infection that was not transcriptionally regulated. Cytoskeletal reorganization was not observed during infection, suggesting that the interaction between actin and E protein has a cell type specific component. PMID:27010925

  14. Differential Occurrence of Interactions and Interaction Domains in Proteins Containing Homopolymeric Amino Acid Repeats.

    PubMed

    Pelassa, Ilaria; Fiumara, Ferdinando

    2015-01-01

    Homopolymeric amino acids repeats (AARs), which are widespread in proteomes, have often been viewed simply as spacers between protein domains, or even as "junk" sequences with no obvious function but with a potential to cause harm upon expansion as in genetic diseases associated with polyglutamine or polyalanine expansions, including Huntington disease and cleidocranial dysplasia. A growing body of evidence indicates however that at least some AARs can form organized, functional protein structures, and can regulate protein function. In particular, certain AARs can mediate protein-protein interactions, either through homotypic AAR-AAR contacts or through heterotypic contacts with other protein domains. It is still unclear however, whether AARs may have a generalized, proteome-wide role in shaping protein-protein interaction networks. Therefore, we have undertaken here a bioinformatics screening of the human proteome and interactome in search of quantitative evidence of such a role. We first identified the sets of proteins that contain repeats of any one of the 20 amino acids, as well as control sets of proteins chosen at random in the proteome. We then analyzed the connectivity between the proteins of the AAR-containing protein sets and we compared it with that observed in the corresponding control networks. We find evidence for different degrees of connectivity in the different AAR-containing protein networks. Indeed, networks of proteins containing polyglutamine, polyglutamate, polyproline, and other AARs show significantly increased levels of connectivity, whereas networks containing polyleucine and other hydrophobic repeats show lower degrees of connectivity. Furthermore, we observed that numerous protein-protein, -nucleic acid, and -lipid interaction domains are significantly enriched in specific AAR protein groups. These findings support the notion of a generalized, combinatorial role of AARs, together with conventional protein interaction domains, in shaping

  15. Investigating Protein-protein Interactions in Live Cells Using Bioluminescence Resonance Energy Transfer

    PubMed Central

    Estruch, Sara B.; Fisher, Simon E.

    2014-01-01

    Assays based on Bioluminescence Resonance Energy Transfer (BRET) provide a sensitive and reliable means to monitor protein-protein interactions in live cells. BRET is the non-radiative transfer of energy from a 'donor' luciferase enzyme to an 'acceptor' fluorescent protein. In the most common configuration of this assay, the donor is Renilla reniformis luciferase and the acceptor is Yellow Fluorescent Protein (YFP). Because the efficiency of energy transfer is strongly distance-dependent, observation of the BRET phenomenon requires that the donor and acceptor be in close proximity. To test for an interaction between two proteins of interest in cultured mammalian cells, one protein is expressed as a fusion with luciferase and the second as a fusion with YFP. An interaction between the two proteins of interest may bring the donor and acceptor sufficiently close for energy transfer to occur. Compared to other techniques for investigating protein-protein interactions, the BRET assay is sensitive, requires little hands-on time and few reagents, and is able to detect interactions which are weak, transient, or dependent on the biochemical environment found within a live cell. It is therefore an ideal approach for confirming putative interactions suggested by yeast two-hybrid or mass spectrometry proteomics studies, and in addition it is well-suited for mapping interacting regions, assessing the effect of post-translational modifications on protein-protein interactions, and evaluating the impact of mutations identified in patient DNA. PMID:24893771

  16. The variable C-terminus of cysteine string proteins modulates exocytosis and protein-protein interactions.

    PubMed

    Boal, Frédéric; Zhang, Hui; Tessier, Céline; Scotti, Pier; Lang, Jochen

    2004-12-28

    Cysteine string proteins (Csps) are vesicle proteins involved in neurotransmission and hormone exocytosis. They are composed of distinct domains: a variable N-terminus, a J-domain followed by a linker region, a cysteine-rich string, and a C-terminus which diverges among isoforms. Their precise function and interactions are not fully understood. Using insulin exocytosis as a model, we show that the linker region and the C-terminus, but not the variable N-terminus, regulate overall secretion. Moreover, endogenous Csp1 binds in a calcium-dependent manner to monomeric VAMP2, and this interaction requires the C-terminus of Csp. The interaction is isoform specific as recombinant Csp1 binds VAMP1 and VAMP7, but not VAMP3. Cross-linking in permeabilized clonal beta-cells revealed homodimerization of Csp which is stimulated by Ca(2+) and again modulated by the variant C-terminus. Our data suggest that both interactions of Csp occur during exocytosis and may explain the effect of the variant C-terminus of this chaperon protein on peptide hormone secretion. PMID:15610015

  17. A Microfluidic Platform for Characterization of Protein—Protein Interactions

    PubMed Central

    Javanmard, Mehdi; Talasaz, Amirali H.; Nemat-Gorgani, Mohsen; Huber, David E.; Pease, Fabian; Ronaghi, Mostafa; Davis, Ronald W.

    2010-01-01

    Traditionally, expensive and time consuming techniques such as mass spectrometry and Western Blotting have been used for characterization of protein–protein interactions. In this paper, we describe the design, fabrication, and testing of a rapid and inexpensive sensor, involving the use of microelectrodes in a microchannel, which can be used for real-time electrical detection of specific interactions between proteins. We have successfully demonstrated detection of target glycoprotein–glycoprotein interactions, antigen-antibody interactions, and glycoprotein-antigen interactions. We have also demonstrated the ability of this technique to distinguish between strong and weak interactions. Using this approach, it may be possible to multiplex an array of these sensors onto a chip and probe a complex mixture for various types of interactions involving protein molecules. PMID:20467571

  18. Predicting Pharmacodynamic Drug-Drug Interactions through Signaling Propagation Interference on Protein-Protein Interaction Networks

    PubMed Central

    Park, Kyunghyun; Kim, Docyong; Ha, Suhyun; Lee, Doheon

    2015-01-01

    As pharmacodynamic drug-drug interactions (PD DDIs) could lead to severe adverse effects in patients, it is important to identify potential PD DDIs in drug development. The signaling starting from drug targets is propagated through protein-protein interaction (PPI) networks. PD DDIs could occur by close interference on the same targets or within the same pathways as well as distant interference through cross-talking pathways. However, most of the previous approaches have considered only close interference by measuring distances between drug targets or comparing target neighbors. We have applied a random walk with restart algorithm to simulate signaling propagation from drug targets in order to capture the possibility of their distant interference. Cross validation with DrugBank and Kyoto Encyclopedia of Genes and Genomes DRUG shows that the proposed method outperforms the previous methods significantly. We also provide a web service with which PD DDIs for drug pairs can be analyzed at http://biosoft.kaist.ac.kr/targetrw. PMID:26469276

  19. Trifunctional cross-linker for mapping protein-protein interaction networks and comparing protein conformational states

    PubMed Central

    Tan, Dan; Li, Qiang; Zhang, Mei-Jun; Liu, Chao; Ma, Chengying; Zhang, Pan; Ding, Yue-He; Fan, Sheng-Bo; Tao, Li; Yang, Bing; Li, Xiangke; Ma, Shoucai; Liu, Junjie; Feng, Boya; Liu, Xiaohui; Wang, Hong-Wei; He, Si-Min; Gao, Ning; Ye, Keqiong; Dong, Meng-Qiu; Lei, Xiaoguang

    2016-01-01

    To improve chemical cross-linking of proteins coupled with mass spectrometry (CXMS), we developed a lysine-targeted enrichable cross-linker containing a biotin tag for affinity purification, a chemical cleavage site to separate cross-linked peptides away from biotin after enrichment, and a spacer arm that can be labeled with stable isotopes for quantitation. By locating the flexible proteins on the surface of 70S ribosome, we show that this trifunctional cross-linker is effective at attaining structural information not easily attainable by crystallography and electron microscopy. From a crude Rrp46 immunoprecipitate, it helped identify two direct binding partners of Rrp46 and 15 protein-protein interactions (PPIs) among the co-immunoprecipitated exosome subunits. Applying it to E. coli and C. elegans lysates, we identified 3130 and 893 inter-linked lysine pairs, representing 677 and 121 PPIs. Using a quantitative CXMS workflow we demonstrate that it can reveal changes in the reactivity of lysine residues due to protein-nucleic acid interaction. DOI: http://dx.doi.org/10.7554/eLife.12509.001 PMID:26952210

  20. Probing Protein-DNA Interactions by Unzipping DNA

    NASA Astrophysics Data System (ADS)

    Wang, Michelle

    2003-03-01

    Protein-DNA interactions are essential to cellular processes. In replication, transcription, recombination, DNA repair, and DNA packaging, proteins bind to DNA as activators or repressors, to recruit other proteins, or to carry out various catalytic activities. I will present Unzipping Force Analysis of Protein Association (UFAPA) as a novel and versatile method for detection of the position and dynamic nature of protein-DNA interactions. A single DNA double helix was unzipped in the presence of DNA-binding proteins using a feedback-enhanced optical trap. When the unzipping fork in a DNA reached a bound protein molecule, we observed a dramatic increase in the tension in the DNA, followed by a sudden tension reduction. Analysis of the unzipping force throughout an unbinding "event" revealed information about the spatial location and dynamic nature of the protein-DNA complex.

  1. Predicting Protein-Protein Interactions from the Molecular to the Proteome Level.

    PubMed

    Keskin, Ozlem; Tuncbag, Nurcan; Gursoy, Attila

    2016-04-27

    Identification of protein-protein interactions (PPIs) is at the center of molecular biology considering the unquestionable role of proteins in cells. Combinatorial interactions result in a repertoire of multiple functions; hence, knowledge of PPI and binding regions naturally serve to functional proteomics and drug discovery. Given experimental limitations to find all interactions in a proteome, computational prediction/modeling of protein interactions is a prerequisite to proceed on the way to complete interactions at the proteome level. This review aims to provide a background on PPIs and their types. Computational methods for PPI predictions can use a variety of biological data including sequence-, evolution-, expression-, and structure-based data. Physical and statistical modeling are commonly used to integrate these data and infer PPI predictions. We review and list the state-of-the-art methods, servers, databases, and tools for protein-protein interaction prediction. PMID:27074302

  2. Manipulating interactions between functional colloidal particles and polyethylene surfaces using interfacial engineering.

    PubMed

    Ziani, Khalid; Barish, Jeffrey A; McClements, David Julian; Goddard, Julie M

    2011-08-01

    The purpose of this study was to examine the interaction between lipid droplets and polyethylene surfaces, representative of those commonly used in food packaging. Lipid droplets with various surface charges were prepared by homogenizing corn oil and water in the presence of surfactants with different electrical characteristics: non-ionic (Tween 80, T80), cationic (lauric arginate, LAE), and/or anionic (sodium dodecyl sulfate, SDS). The ionic properties of polyethylene surfaces were modified by UV-treatment. Stable emulsions containing small droplets (d<200 nm) with nearly neutral (T80), cationic (T80: LAE), and anionic (T80: SDS) charges were prepared by adding different levels of the ionic surfactants to Tween 80 stabilized emulsions. Scanning electronic microscopy (SEM), confocal fluorescence microscopy, and ATR-FTIR showed that the number of droplets attached to the polyethylene surfaces depended on the droplet charge and the polyethylene surface characteristics. The greatest degree of droplet adsorption was observed for the cationic droplets to the UV-ozone treated polyethylene surfaces, which was attributed to electrostatic attraction. These results are important for understanding the behavior of encapsulated lipophilic components in food containers. PMID:21555134

  3. Spin manipulation in carbon nanotubes: All electrical spin filtering through spin-orbit interactions

    NASA Astrophysics Data System (ADS)

    Diniz, G. S.; Latgé, A.; Ulloa, S. E.

    2011-03-01

    Carbon nanotubes (CNTs) are known to exhibit interesting physical properties, such as metallic or insulating behavior for different chiral vectors. Application of external electric fields and the presence of spin-orbit interaction (SOI) result in modification of the energy level structure of CNTs and their conductance profiles. SOI couples spin and orbital degrees of freedom in these nanostructures, and we explore this effect in this work. We present calculations of the electronic transport of different single-wall CNTs in the presence of SOI. Our calculation uses a single-orbital tight-binding Hamiltonian representation and the equilibrium surface Green's function formalism to calculate electronic transport. We consider the effects of both Rashba and intrinsic SOIs. Our results show possible implementations of carbon nanotubes as spin filtering devices for spatially asymmetric electric fields. We further discuss the spin polarization for different CNT size, chirality, field strength, and the spatially varying fields induced by the adsorption of DNA on their surface. Supported by NSF and CAPES/Fulbright.

  4. A novel method for protein-protein interaction site prediction using phylogenetic substitution models

    PubMed Central

    La, David; Kihara, Daisuke

    2011-01-01

    Protein-protein binding events mediate many critical biological functions in the cell. Typically, functionally important sites in proteins can be well identified by considering sequence conservation. However, protein-protein interaction sites exhibit higher sequence variation than other functional regions, such as catalytic sites of enzymes. Consequently, the mutational behavior leading to weak sequence conservation poses significant challenges to the protein-protein interaction site prediction. Here, we present a phylogenetic framework to capture critical sequence variations that favor the selection of residues essential for protein-protein binding. Through the comprehensive analysis of diverse protein families, we show that protein binding interfaces exhibit distinct amino acid substitution as compared with other surface residues. Based on this analysis, we have developed a novel method, BindML, which utilizes the substitution models to predict protein-protein binding sites of protein with unknown interacting partners. BindML estimates the likelihood that a phylogenetic tree of a local surface region in a query protein structure follows the substitution patterns of protein binding interface and non-binding surfaces. BindML is shown to perform well compared to alternative methods for protein binding interface prediction. The methodology developed in this study is very versatile in the sense that it can be generally applied for predicting other types of functional sites, such as DNA, RNA, and membrane binding sites in proteins. PMID:21989996

  5. Manipulation of the local density of photonic states to elucidate fluorescent protein emission rates.

    PubMed

    Cesa, Yanina; Blum, Christian; van den Broek, Johanna M; Mosk, Allard P; Vos, Willem L; Subramaniam, Vinod

    2009-04-14

    We present experiments to determine the quantum efficiency and emission oscillator strength of exclusively the emitting states of the widely used enhanced green fluorescent protein (EGFP). We positioned the emitters at precisely defined distances from a mirror to control the local density of optical states, resulting in characteristic changes in the fluorescence decay rate that we monitored by fluorescence lifetime microscopy. To the best of our knowledge, this is the first emission lifetime control of a biological emitter. From the oscillation of the observed emission lifetimes as a function of the emitter to mirror distance, we determined the radiative and nonradiative decay rates of the fluorophore. Since only the emitting species contribute to the change in emission lifetimes, the rates determined characterize specifically the quantum efficiency and oscillator strength of the on-states of the emitter, in contrast to other methods that do not differentiate between emitting and dark states. The method reported is especially interesting for photophysically complex systems like fluorescent proteins, where a range of emitting and dark forms has been observed. We have validated the analysis method using Rhodamine 6G dye, obtaining results in very good agreement with the literature. For EGFP we determine the quantum efficiency of the on-states to be 72%. As expected for this complex system, our value is higher than that determined by methods that average over on- and off-states. PMID:19325987

  6. BiopLib and BiopTools—a C programming library and toolset for manipulating protein structure

    PubMed Central

    Porter, Craig T.; Martin, Andrew C.R.

    2015-01-01

    Summary: We describe BiopLib, a mature C programming library for manipulating protein structure, and BiopTools, a set of command-line tools which exploit BiopLib. The library also provides a small number of functions for handling protein sequence and general purpose programming and mathematics. BiopLib transparently handles PDBML (XML) format and standard PDB files. BiopTools provides facilities ranging from renumbering atoms and residues to calculation of solvent accessibility. Availability and implementation: BiopLib and BiopTools are implemented in standard ANSI C. The core of the BiopLib library is a reliable PDB parser that handles alternate occupancies and deals with compressed PDB files and PDBML files automatically. The library is designed to be as flexible as possible, allowing users to handle PDB data as a simple list of atoms, or in a structured form using chains, residues and atoms. Many of the BiopTools command-line tools act as filters, taking a PDB (or PDBML) file as input and producing a PDB (or PDBML) file as output. All code is open source and documented using Doxygen. It is provided under the GNU Public Licence and is available from the authors’ web site or from GitHub. Contact: andrew@bioinf.org.uk PMID:26323716

  7. Network representation of protein interactions-Experimental results.

    PubMed

    Kurzbach, Dennis; Flamm, Andrea G; Sára, Tomáš

    2016-09-01

    A graph theoretical analysis of nuclear magnetic resonance (NMR) data of six different protein interactions has been presented. The representation of the protein interaction data as a graph or network reveals that all of the studied interactions are based on a common functional concept. They all involve a single densely packed hub of functionally correlated residues that mediate the ligand binding events. This is found independent of the kind of protein (folded or unfolded) or ligand (protein, polymer or small molecule). Furthermore, the power of the graph analysis is demonstrated at the examples of the Calmodulin (CaM)/Calcium and the Cold Shock Protein A (CspA)/RNA interaction. The presented approach enables the precise determination of multiple binding sites for the respective ligand molecules. PMID:27272395

  8. Single Molecule Approaches in RNA-Protein Interactions.

    PubMed

    Serebrov, Victor; Moore, Melissa J

    2016-01-01

    RNA-protein interactions govern every aspect of RNA metabolism, and aberrant RNA-binding proteins are the cause of hundreds of genetic diseases. Quantitative measurements of these interactions are necessary in order to understand mechanisms leading to diseases and to develop efficient therapies. Existing methods of RNA-protein interactome capture can afford a comprehensive snapshot of RNA-protein interaction networks but lack the ability to characterize the dynamics of these interactions. As all ensemble methods, their resolution is also limited by statistical averaging. Here we discuss recent advances in single molecule techniques that have the potential to tackle these challenges. We also provide a thorough overview of single molecule colocalization microscopy and the essential protein and RNA tagging and detection techniques. PMID:27256383

  9. [Multiparticle computer simulation of protein interactions in the photosynthetic membrane].

    PubMed

    Riznichenko, G Iu; Kovalenko, I B; Abaturova, A M; D'iakonova, A N; Kniazeva, O S; Ustinin, D M; Khrushchev, S S; Rubin, A B

    2011-01-01

    The basic principles of the design of direct multiparticle models and the results of multiparticle computer simulation of electron transfer by mobile protein carriers in the photosynthetic membrane of a chloroplast thylakoid are presented. The reactions of complex formation of the protein plastocyanin with the protein cytochrome f and the pigment-protein complex of photosystem I, as well as of the protein ferredoxin with the protein FNR and photosystem 1 are considered. The role of diffusion and electrostatic interactions is discussed, and the effect of the shape of the reaction volume and ionic strength on the rate of electron transport are discussed. PMID:22117434

  10. Domain-mediated protein interaction prediction: From genome to network.

    PubMed

    Reimand, Jüri; Hui, Shirley; Jain, Shobhit; Law, Brian; Bader, Gary D

    2012-08-14

    Protein-protein interactions (PPIs), involved in many biological processes such as cellular signaling, are ultimately encoded in the genome. Solving the problem of predicting protein interactions from the genome sequence will lead to increased understanding of complex networks, evolution and human disease. We can learn the relationship between genomes and networks by focusing on an easily approachable subset of high-resolution protein interactions that are mediated by peptide recognition modules (PRMs) such as PDZ, WW and SH3 domains. This review focuses on computational prediction and analysis of PRM-mediated networks and discusses sequence- and structure-based interaction predictors, techniques and datasets for identifying physiologically relevant PPIs, and interpreting high-resolution interaction networks in the context of evolution and human disease. PMID:22561014

  11. Aromatic-aromatic interactions: analysis of π-π interactions in interleukins and TNF proteins

    PubMed Central

    Sivasakthi, Vaideeswaran; Anitha, Parimelzaghan; Kumar, Kalavathi Murugan; Bag, Susmita; Senthilvel, Padmanaban; Lavanya, Pandian; Swetha, Rayapadi; Anbarasu, Anand; Ramaiah, Sudha

    2013-01-01

    Aromatic-aromatic hydrogen bonds are important in many areas of chemistry, biology and materials science. In this study we have analyzed the roles played by the π-π interactions in interleukins (ILs) and tumor necrosis factor (TNF) proteins. Majority of π-π interacting residues are conserved in ILs and TNF proteins. The accessible surface area calculations in these proteins reveal that these interactions might be important in stabilizing the inner core regions of these proteins. In addition to π-π interactions, the aromatic residues also form π-networks in ILs and TNF proteins. The results obtained in the present study indicate that π-π interactions and π-π networks play important roles in the structural stability of ILs and TNF proteins. PMID:23750094

  12. Porcine reproductive and respiratory syndrome virus envelope (E) protein interacts with mitochondrial proteins and induces apoptosis.

    PubMed

    Pujhari, Sujit; Zakhartchouk, Alexander N

    2016-07-01

    Porcine reproductive and respiratory syndrome virus (PRRSV) causes significant economic losses for the swine industry worldwide. The PRRSV E protein, encoded by ORF 2b, is one of the non-glycosylated minor structural proteins. In this study, we present evidence for the interaction of the E protein with mitochondrial proteins ATP5A (part of ATP synthase complex), prohibitin, and ADP/ATP translocase. We additionally demonstrate partial mitochondrial localization of the E protein in transfected cells. To functionally investigate these interactions, we infected MARC-145 cells with PRRSV or alphavirus replicon particles (VRPs) expressing PRRSV E protein. In infected cells, production of ATP was significantly reduced. The E protein also induced apoptosis by activating caspase-3, which results in PARP cleavage. Taken together, these data suggest that the PRRSV E protein interacts with mitochondrial proteins and induces apoptosis by inhibiting ATP production. PMID:27068165

  13. A proteome-wide protein interaction map for Campylobacter jejuni

    PubMed Central

    Parrish, Jodi R; Yu, Jingkai; Liu, Guozhen; Hines, Julie A; Chan, Jason E; Mangiola, Bernie A; Zhang, Huamei; Pacifico, Svetlana; Fotouhi, Farshad; DiRita, Victor J; Ideker, Trey; Andrews, Phillip; Finley, Russell L

    2007-01-01

    Background Data from large-scale protein interaction screens for humans and model eukaryotes have been invaluable for developing systems-level models of biological processes. Despite this value, only a limited amount of interaction data is available for prokaryotes. Here we report the systematic identification of protein interactions for the bacterium Campylobacter jejuni, a food-borne pathogen and a major cause of gastroenteritis worldwide. Results Using high-throughput yeast two-hybrid screens we detected and reproduced 11,687 interactions. The resulting interaction map includes 80% of the predicted C. jejuni NCTC11168 proteins and places a large number of poorly characterized proteins into networks that provide initial clues about their functions. We used the map to identify a number of conserved subnetworks by comparison to protein networks from Escherichia coli and Saccharomyces cerevisiae. We also demonstrate the value of the interactome data for mapping biological pathways by identifying the C. jejuni chemotaxis pathway. Finally, the interaction map also includes a large subnetwork of putative essential genes that may be used to identify potential new antimicrobial drug targets for C. jejuni and related organisms. Conclusion The C. jejuni protein interaction map is one of the most comprehensive yet determined for a free-living organism and nearly doubles the binary interactions available for the prokaryotic kingdom. This high level of coverage facilitates pathway mapping and function prediction for a large number of C. jejuni proteins as well as orthologous proteins from other organisms. The broad coverage also facilitates cross-species comparisons for the identification of evolutionarily conserved subnetworks of protein interactions. PMID:17615063

  14. Direct Manipulation Interfaces.

    ERIC Educational Resources Information Center

    Hutchins, Edwin L.; And Others

    This paper presents a cognitive account of both the advantages and disadvantages of direct manipulation interfaces, i.e., the use of icons to manipulate and interact directly with data rather than writing programs or calling on a set of statistical subroutines. Two underlying phenomena that give rise to the sensation of directness are identified.…

  15. Revisiting Mathematics Manipulative Materials

    ERIC Educational Resources Information Center

    Swan, Paul; Marshall, Linda

    2010-01-01

    It is over 12 years since "APMC" published Bob Perry and Peter Howard's research on the use of mathematics manipulative materials in primary mathematics classrooms. Since then the availability of virtual manipulatives and associated access to computers and interactive whiteboards have caused educators to rethink the use of mathematics manipulative…

  16. Visualization of Host-Polerovirus Interaction Topologies Using Protein Interaction Reporter Technology

    PubMed Central

    DeBlasio, Stacy L.; Chavez, Juan D.; Alexander, Mariko M.; Ramsey, John; Eng, Jimmy K.; Mahoney, Jaclyn; Gray, Stewart M.; Bruce, James E.

    2015-01-01

    ABSTRACT Demonstrating direct interactions between host and virus proteins during infection is a major goal and challenge for the field of virology. Most protein interactions are not binary or easily amenable to structural determination. Using infectious preparations of a polerovirus (Potato leafroll virus [PLRV]) and protein interaction reporter (PIR), a revolutionary technology that couples a mass spectrometric-cleavable chemical cross-linker with high-resolution mass spectrometry, we provide the first report of a host-pathogen protein interaction network that includes data-derived, topological features for every cross-linked site that was identified. We show that PLRV virions have hot spots of protein interaction and multifunctional surface topologies, revealing how these plant viruses maximize their use of binding interfaces. Modeling data, guided by cross-linking constraints, suggest asymmetric packing of the major capsid protein in the virion, which supports previous epitope mapping studies. Protein interaction topologies are conserved with other species in the Luteoviridae and with unrelated viruses in the Herpesviridae and Adenoviridae. Functional analysis of three PLRV-interacting host proteins in planta using a reverse-genetics approach revealed a complex, molecular tug-of-war between host and virus. Structural mimicry and diversifying selection—hallmarks of host-pathogen interactions—were identified within host and viral binding interfaces predicted by our models. These results illuminate the functional diversity of the PLRV-host protein interaction network and demonstrate the usefulness of PIR technology for precision mapping of functional host-pathogen protein interaction topologies. IMPORTANCE The exterior shape of a plant virus and its interacting host and insect vector proteins determine whether a virus will be transmitted by an insect or infect a specific host. Gaining this information is difficult and requires years of experimentation. We used

  17. Discovering novel protein-protein interactions by measuring the protein semantic similarity from the biomedical literature.

    PubMed

    Chiang, Jung-Hsien; Ju, Jiun-Huang

    2014-12-01

    Protein-protein interactions (PPIs) are involved in the majority of biological processes. Identification of PPIs is therefore one of the key aims of biological research. Although there are many databases of PPIs, many other unidentified PPIs could be buried in the biomedical literature. Therefore, automated identification of PPIs from biomedical literature repositories could be used to discover otherwise hidden interactions. Search engines, such as Google, have been successfully applied to measure the relatedness among words. Inspired by such approaches, we propose a novel method to identify PPIs through semantic similarity measures among protein mentions. We define six semantic similarity measures as features based on the page counts retrieved from the MEDLINE database. A machine learning classifier, Random Forest, is trained using the above features. The proposed approach achieve an averaged micro-F of 71.28% and an averaged macro-F of 64.03% over five PPI corpora, an improvement over the results of using only the conventional co-occurrence feature (averaged micro-F of 68.79% and an averaged macro-F of 60.49%). A relation-word reinforcement further improves the averaged micro-F to 71.3% and averaged macro-F to 65.12%. Comparing the results of the current work with other studies on the AIMed corpus (ranging from 77.58% to 85.1% in micro-F, 62.18% to 76.27% in macro-F), we show that the proposed approach achieves micro-F of 81.88% and macro-F of 64.01% without the use of sophisticated feature extraction. Finally, we manually examine the newly discovered PPI pairs based on a literature review, and the results suggest that our approach could extract novel protein-protein interactions. PMID:25385082

  18. The origins of the evolutionary signal used to predict protein-protein interactions

    PubMed Central

    2012-01-01

    Background The correlation of genetic distances between pairs of protein sequence alignments has been used to infer protein-protein interactions. It has been suggested that these correlations are based on the signal of co-evolution between interacting proteins. However, although mutations in different proteins associated with maintaining an interaction clearly occur (particularly in binding interfaces and neighbourhoods), many other factors contribute to correlated rates of sequence evolution. Proteins in the same genome are usually linked by shared evolutionary history and so it would be expected that there would be topological similarities in their phylogenetic trees, whether they are interacting or not. For this reason the underlying species tree is often corrected for. Moreover processes such as expression level, are known to effect evolutionary rates. However, it has been argued that the correlated rates of evolution used to predict protein interaction explicitly includes shared evolutionary history; here we test this hypothesis. Results In order to identify the evolutionary mechanisms giving rise to the correlations between interaction proteins, we use phylogenetic methods to distinguish similarities in tree topologies from similarities in genetic distances. We use a range of datasets of interacting and non-interacting proteins from Saccharomyces cerevisiae. We find that the signal of correlated evolution between interacting proteins is predominantly a result of shared evolutionary rates, rather than similarities in tree topology, independent of evolutionary divergence. Conclusions Since interacting proteins do not have tree topologies that are more similar than the control group of non-interacting proteins, it is likely that coevolution does not contribute much to, if any, of the observed correlations. PMID:23217198

  19. Composition-Gradient Static Light Scattering and the Quantification of Biomolecular Interactions in Therapeutic Proteins

    NASA Astrophysics Data System (ADS)

    Some, Daniel

    2010-03-01

    Macromolecular interactions of interest to the pharmaceutical industry cover a variety of phenomena: binding of proteins to form well-defined complexes; reversible and irreversible oligomerization; and non-specific intermolecular interactions. The analysis and manipulation of these phenomena are crucial to the successful development, manufacture, storage and delivery of biological drugs such as antibodies. Light scattering (LS) has proven to be one of the most versatile free-solution and label-free methods for studying proteins and their interactions. Previously limited primarily to assessing molar mass, size and oligomerization state, the recent emergence of automated Composition-Gradient Static Light Scattering (Attri, A.; Minton, A.P. Anal. Biochem. 2005; 346(1), 132--8), or CG-SLS, extends the range of biotech LS applications to equilibrium binding affinity and stoichiometry of bound complexes, kinetics of association and dissociation, and non-specific interactions (attractive and repulsive). In this talk I present progress in CG-SLS for biophysical characterization of pharmaceutical protein-protein interactions. In the drug development phase, CG-SLS studies of antibody-antigen complexes compliments other biomolecular interaction techniques commonly found in the biotech world such as surface plasmon resonance (SPR). In the formulation development stage, long-term stability of drug product is sought. Protein degradation modes include irreversible aggregation, which may lead to adverse physiological effects, and reversible self-association, which affects solution viscosity and hence injectable drug delivery. CG-SLS addresses both of these, the former via determination of virial coefficients, which describe the overall non-specific attraction or repulsion between molecules and may be used to optimize the formulation buffer to minimize aggregation, and the latter by binding affinity and stoichiometry of the associated complexes.

  20. Visualization of electron transfer interactions of membrane proteins

    NASA Astrophysics Data System (ADS)

    Kawato, Suguru

    1991-08-01

    To visualize electron transfer interactions of proteins in the cellular nieinbrane, we have developed a polarized laser flash-induced anisotropy decay imaging. The time-resolved anisotropy is particularly sensitive to protein-protein interactions. This technique has been successfully applied to examine formation and dissociation of electron transfer complex in adrenal cortex and liver. Electron transfer plays a significant role for steroid hormone synthesis from cholesterol in adrenalcortex and for drug metabolism in liver such as detoxification of chemical compounds. Several redox partners perticipate in dynamic electron transfer interactions. The terminal enzyme cytochrome P-450 receives electrons to activate molecular oxygen, resulting in hydroxylation of various substrates.

  1. 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

  2. Protein solvent and weak protein protein interactions in halophilic malate dehydrogenase

    NASA Astrophysics Data System (ADS)

    Ebel, Christine; Faou, Pierre; Zaccai, Giuseppe

    1999-01-01

    With the aim to correlate the solvation, stability and solubility properties of halophilic malate dehydrogenase, we characterized its weak interparticle interactions by small-angle neutron scattering in various solvents. The protein concentration dependence of the apparent radius of gyration and forward scattered intensity extrapolated from Guinier plots, and thus the second virial coefficient, A2, were determined for each solvent condition. In NaCl 1M+2-methylpentane-2,4-diol 30%, a solvent that promotes protein crystallization, A2 is negative, -0.4×10 -4 ml mol g -2 and indicating attractive interactions; in ammonium sulfate 3M, in which the protein precipitates at high concentrations, A2˜0. In 2-5M NaCl, 1-3.5M NaOAc, 1-4.5M KF or 1-2M (NH 4) 2SO 4, in which the protein is very soluble, A2 is positive with a value of the order of 0.4×10 -4 ml mol g -2 which decreases with increasing salt concentration. In MgCl 2 however, A2 increases with increasing salt concentration from 0.2 to 1.3M.

  3. Protein-Protein Interactions in high moisture-extruded meat analogs and heat-induce soy Protein Gels

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Two commercial soy protein isolates were made into fibrous meat analogs by high moisture extrusion or into gels by heating and cooling, at varying concentrations and/or temperatures. Protein-protein interactions by extrusion or gelation were investigated through protein solubility studies of raw an...

  4. Template-based structure modeling of protein-protein interactions

    PubMed Central

    Szilagyi, Andras; Zhang, Yang

    2014-01-01

    The structure of protein-protein complexes can be constructed by using the known structure of other protein complexes as a template. The complex structure templates are generally detected either by homology-based sequence alignments or, given the structure of monomer components, by structure-based comparisons. Critical improvements have been made in recent years by utilizing interface recognition and by recombining monomer and complex template libraries. Encouraging progress has also been witnessed in genome-wide applications of template-based modeling, with modeling accuracy comparable to high-throughput experimental data. Nevertheless, bottlenecks exist due to the incompleteness of the proteinprotein complex structure library and the lack of methods for distant homologous template identification and full-length complex structure refinement. PMID:24721449

  5. Bilayer-thickness-mediated interactions between integral membrane proteins.

    PubMed

    Kahraman, Osman; Koch, Peter D; Klug, William S; Haselwandter, Christoph A

    2016-04-01

    Hydrophobic thickness mismatch between integral membrane proteins and the surrounding lipid bilayer can produce lipid bilayer thickness deformations. Experiment and theory have shown that protein-induced lipid bilayer thickness deformations can yield energetically favorable bilayer-mediated interactions between integral membrane proteins, and large-scale organization of integral membrane proteins into protein clusters in cell membranes. Within the continuum elasticity theory of membranes, the energy cost of protein-induced bilayer thickness deformations can be captured by considering compression and expansion of the bilayer hydrophobic core, membrane tension, and bilayer bending, resulting in biharmonic equilibrium equations describing the shape of lipid bilayers for a given set of bilayer-protein boundary conditions. Here we develop a combined analytic and numerical methodology for the solution of the equilibrium elastic equations associated with protein-induced lipid bilayer deformations. Our methodology allows accurate prediction of thickness-mediated protein interactions for arbitrary protein symmetries at arbitrary protein separations and relative orientations. We provide exact analytic solutions for cylindrical integral membrane proteins with constant and varying hydrophobic thickness, and develop perturbative analytic solutions for noncylindrical protein shapes. We complement these analytic solutions, and assess their accuracy, by developing both finite element and finite difference numerical solution schemes. We provide error estimates of our numerical solution schemes and systematically assess their convergence properties. Taken together, the work presented here puts into place an analytic and numerical framework which allows calculation of bilayer-mediated elastic interactions between integral membrane proteins for the complicated protein shapes suggested by structural biology and at the small protein separations most relevant for the crowded membrane

  6. Bilayer-thickness-mediated interactions between integral membrane proteins

    NASA Astrophysics Data System (ADS)

    Kahraman, Osman; Koch, Peter D.; Klug, William S.; Haselwandter, Christoph A.

    2016-04-01

    Hydrophobic thickness mismatch between integral membrane proteins and the surrounding lipid bilayer can produce lipid bilayer thickness deformations. Experiment and theory have shown that protein-induced lipid bilayer thickness deformations can yield energetically favorable bilayer-mediated interactions between integral membrane proteins, and large-scale organization of integral membrane proteins into protein clusters in cell membranes. Within the continuum elasticity theory of membranes, the energy cost of protein-induced bilayer thickness deformations can be captured by considering compression and expansion of the bilayer hydrophobic core, membrane tension, and bilayer bending, resulting in biharmonic equilibrium equations describing the shape of lipid bilayers for a given set of bilayer-protein boundary conditions. Here we develop a combined analytic and numerical methodology for the solution of the equilibrium elastic equations associated with protein-induced lipid bilayer deformations. Our methodology allows accurate prediction of thickness-mediated protein interactions for arbitrary protein symmetries at arbitrary protein separations and relative orientations. We provide exact analytic solutions for cylindrical integral membrane proteins with constant and varying hydrophobic thickness, and develop perturbative analytic solutions for noncylindrical protein shapes. We complement these analytic solutions, and assess their accuracy, by developing both finite element and finite difference numerical solution schemes. We provide error estimates of our numerical solution schemes and systematically assess their convergence properties. Taken together, the work presented here puts into place an analytic and numerical framework which allows calculation of bilayer-mediated elastic interactions between integral membrane proteins for the complicated protein shapes suggested by structural biology and at the small protein separations most relevant for the crowded membrane

  7. Monitoring single membrane protein dynamics in a liposome manipulated in solution by the ABELtrap

    NASA Astrophysics Data System (ADS)

    Rendler, T.; Renz, M.; Hammann, E.; Ernst, S.; Zarrabi, N.; Börsch, M.

    2011-02-01

    FoF1-ATP synthase is the essential membrane enzyme maintaining the cellular level of adenosine triphosphate (ATP) and comprises two rotary motors. We measure subunit rotation in FoF1-ATP synthase by intramolecular Foerster resonance energy transfer (FRET) between two fluorophores at the rotor and at the stator of the enzyme. Confocal FRET measurements of freely diffusing single enzymes in lipid vesicles are limited to hundreds of milliseconds by the transit times through the laser focus. We evaluate two different methods to trap the enzyme inside the confocal volume in order to extend the observation times. Monte Carlo simulations show that optical tweezers with low laser power are not suitable for lipid vesicles with a diameter of 130 nm. A. E. Cohen (Harvard) and W. E. Moerner (Stanford) have recently developed an Anti-Brownian electrokinetic trap (ABELtrap) which is capable to apparently immobilize single molecules, proteins, viruses or vesicles in solution. Trapping of fluorescent particles is achieved by applying a real time, position-dependent feedback to four electrodes in a microfluidic device. The standard deviation from a given target position in the ABELtrap is smaller than 200 nm. We develop a combination of the ABELtrap with confocal FRET measurements to monitor single membrane enzyme dynamics by FRET for more than 10 seconds in solution.

  8. Global approaches to study protein-protein interactions among viruses and hosts.

    PubMed

    Mendez-Rios, Jorge; Uetz, Peter

    2010-02-01

    While high-throughput protein-protein interaction screens were first published approximately 10 years ago, systematic attempts to map interactions among viruses and hosts started only a few years ago. HIV-human interactions dominate host-pathogen interaction databases (with approximately 2000 interactions) despite the fact that probably none of these interactions have been identified in systematic interaction screens. Recently, combinations of protein interaction data with RNAi and other functional genomics data allowed researchers to model more complex interaction networks. The rapid progress in this area promises a flood of new data in the near future, with clinical applications as soon as structural and functional genomics catches up with next-generation sequencing of human variation and structure-based drug design. PMID:20143950

  9. Detecting Protein-Protein Interactions in Vesicular Stomatitis Virus Using a Cytoplasmic Yeast Two Hybrid System

    PubMed Central

    Moerdyk-Schauwecker, Megan; DeStephanis, Darla; Hastie, Eric; Grdzelishvili, Valery Z.

    2011-01-01

    Summary Protein-protein interactions play an important role in many virus-encoded functions and in virus-host interactions. While a “classical” yeast two-hybrid system (Y2H) is one of the most common techniques to detect such interactions, it has a number of limitations, including a requirement for the proteins of interest to be relocated to the nucleus. Modified Y2H, such as the Sos recruitment system (SRS), which detect interactions occurring in the cytoplasm rather than the nucleus, allow proteins from viruses replicating in the cytoplasm to be tested in a more natural context. In this study, a SRS was used to detect interactions involving proteins from vesicular stomatitis virus (VSV), a prototypic non-segmented negative strand RNA (NNS) virus. All five full-length VSV proteins, as well as several truncated proteins, were screened against each other. Using the SRS, most interactions demonstrated previously involving VSV phosphoprotein, nucleocapsid (N) and large polymerase proteins were confirmed independently, while difficulties were encountered using the membrane associated matrix and glycoproteins. A human cDNA library was also screened against VSV N protein and one cellular protein, SFRS18, was identified which interacted with N in this context. The system presented can be redesigned easily for studies in other less tractable NNS viruses. PMID:21320532

  10. Choosing negative examples for the prediction of protein-protein interactions

    PubMed Central

    Ben-Hur, Asa; Noble, William Stafford

    2006-01-01

    The protein-protein interaction networks of even well-studied model organisms are sketchy at best, highlighting the continued need for computational methods to help direct experimentalists in the search for novel interactions. This need has prompted the development of a number of methods for predicting protein-protein interactions based on various sources of data and methodologies. The common method for choosing negative examples for training a predictor of protein-protein interactions is based on annotations of cellular localization, and the observation that pairs of proteins that have different localization patterns are unlikely to interact. While this method leads to high quality sets of non-interacting proteins, we find that this choice can lead to biased estimates of prediction accuracy, because the constraints placed on the distribution of the negative examples makes the task easier. The effects of this bias are demonstrated in the context of both sequence-based and non-sequence based features used for predicting protein-protein interactions. PMID:16723005

  11. A method for investigating protein-protein interactions related to Salmonella typhimurium pathogenesis

    SciTech Connect

    Chowdhury, Saiful M.; Shi, Liang; Yoon, Hyunjin; Ansong, Charles; Rommereim, Leah M.; Norbeck, Angela D.; Auberry, Kenneth J.; Moore, R. J.; Adkins, Joshua N.; Heffron, Fred; Smith, Richard D.

    2009-02-10

    We successfully modified an existing method to investigate protein-protein interactions in the pathogenic bacterium Salmonella typhimurium (STM). This method includes i) addition of a histidine-biotin-histidine tag to the bait proteins via recombinant DNA techniques; ii) in vivo cross-linking with formaldehyde; iii) tandem affinity purification of bait proteins under fully denaturing conditions; and iv) identification of the proteins cross-linked to the bait proteins by liquid-chromatography in conjunction with tandem mass-spectrometry. In vivo cross-linking stabilized protein interactions permitted the subsequent two-step purification step conducted under denaturing conditions. The two-step purification greatly reduced nonspecific binding of non-cross-linked proteins to bait proteins. Two different negative controls were employed to reduce false-positive identification. In an initial demonstration of this approach, we tagged three selected STM proteins- HimD, PduB and PhoP- with known binding partners that ranged from stable (e.g., HimD) to transient (i.e., PhoP). Distinct sets of interacting proteins were identified with each bait protein, including the known binding partners such as HimA for HimD, as well as anticipated and unexpected binding partners. Our results suggest that novel protein-protein interactions may be critical to pathogenesis by Salmonella typhimurium. .

  12. PPISEARCHENGINE: gene ontology-based search for protein-protein interactions.

    PubMed

    Park, Byungkyu; Cui, Guangyu; Lee, Hyunjin; Huang, De-Shuang; Han, Kyungsook

    2013-01-01

    This paper presents a new search engine called PPISearchEngine which finds protein-protein interactions (PPIs) using the gene ontology (GO) and the biological relations of proteins. For efficient retrieval of PPIs, each GO term is assigned a prime number and the relation between the terms is represented by the product of prime numbers. This representation is hidden from users but facilitates the search for the interactions of a query protein by unique prime factorisation of the number that represents the query protein. For a query protein, PPISearchEngine considers not only the GO term associated with the query protein but also the GO terms at the lower level than the GO term in the GO hierarchy, and finds all the interactions of the query protein which satisfy the search condition. In contrast, the standard keyword-matching or ID-matching search method cannot find the interactions of a protein unless the interactions involve a protein with explicit annotations. To the best of our knowledge, this search engine is the first method that can process queries like 'for protein p with GO [Formula: see text], find p's interaction partners with GO [Formula: see text]'. PPISearchEngine is freely available to academics at http://search.hpid.org/. PMID:22316075

  13. Identification of brain-specific angiogenesis inhibitor 2 as an interaction partner of glutaminase interacting protein

    SciTech Connect

    Zencir, Sevil; Ovee, Mohiuddin; Dobson, Melanie J.; Banerjee, Monimoy; Topcu, Zeki; Mohanty, Smita

    2011-08-12

    Highlights: {yields} Brain-specific angiogenesis inhibitor 2 (BAI2) is a new partner protein for GIP. {yields} BAI2 interaction with GIP was revealed by yeast two-hybrid assay. {yields} Binding of BAI2 to GIP was characterized by NMR, CD and fluorescence. {yields} BAI2 and GIP binding was mediated through the C-terminus of BAI2. -- Abstract: The vast majority of physiological processes in living cells are mediated by protein-protein interactions often specified by particular protein sequence motifs. PDZ domains, composed of 80-100 amino acid residues, are an important class of interaction motif. Among the PDZ-containing proteins, glutaminase interacting protein (GIP), also known as Tax Interacting Protein TIP-1, is unique in being composed almost exclusively of a single PDZ domain. GIP has important roles in cellular signaling, protein scaffolding and modulation of tumor growth and interacts with a number of physiological partner proteins, including Glutaminase L, {beta}-Catenin, FAS, HTLV-1 Tax, HPV16 E6, Rhotekin and Kir 2.3. To identify the network of proteins that interact with GIP, a human fetal brain cDNA library was screened using a yeast two-hybrid assay with GIP as bait. We identified brain-specific angiogenesis inhibitor 2 (BAI2), a member of the adhesion-G protein-coupled receptors (GPCRs), as a new partner of GIP. BAI2 is expressed primarily in neurons, further expanding GIP cellular functions. The interaction between GIP and the carboxy-terminus of BAI2 was characterized using fluorescence, circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopy assays. These biophysical analyses support the interaction identified in the yeast two-hybrid assay. This is the first study reporting BAI2 as an interaction partner of GIP.

  14. Manipulation of Goos-Hänchen shifts in the atomic configuration of mercury via interacting dark-state resonances

    NASA Astrophysics Data System (ADS)

    Hamedi, H. R.; Radmehr, Arash; Sahrai, M.

    2014-11-01

    We study the manipulation of Goos-Hänchen (GH) shifts for the reflected and transmitted probe light pulses injected into a cavity containing four-level configuration mercury atoms where the probe transition is in the ultraviolet (UV) region with a wavelength of 253.7 nm . Different behaviors of the GH shifts can be observed in the absence, or presence, of two driving fields as well as an incoherent pump field. When neither coherent driving fields nor incoherent pumping is turned on, we realize negative reflected GH shifts for anomalous dispersion. Including only one driving field leads to subluminal-based light propagation with positive lateral shifts at certain incident angles. Taking into account the impact of both driving fields, negative GH shifts reappear in the reflected part of the incident light. The origin of this defect is attributed to interacting double dark resonances due to a high-resolution absorption peaks with a very steep negative slope of dispersion in the susceptibility profile. We then show that one can surpass this defect by applying a weak incoherent pumping field to obtain positive GH shifts for both reflected and transmitted light beams. Finally, using the 6 1P1↔6 1S0 transition of Hg, we generalize our study to the case where the wavelength of the probe transition is 185 nm which is in the vacuum-ultraviolet domain. Although the number of oscillations is now increased, however, similar results are reported with respect to the case of UV transition.

  15. Magnetic Radial Vortex Stabilization and Efficient Manipulation Driven by the Dzyaloshinskii-Moriya Interaction and Spin-Transfer Torque.

    PubMed

    Siracusano, G; Tomasello, R; Giordano, A; Puliafito, V; Azzerboni, B; Ozatay, O; Carpentieri, M; Finocchio, G

    2016-08-19

    Solitons are very promising for the design of the next generation of ultralow power devices for storage and computation. The key ingredient to achieving this goal is the fundamental understanding of their stabilization and manipulation. Here, we show how the interfacial Dzyaloshinskii-Moriya Interaction (IDMI) is able to lift the energy degeneracy of a magnetic vortex state by stabilizing a topological soliton with radial chirality, hereafter called radial vortex. It has a noninteger Skyrmion number S (0.5<|S|<1) due to both the vortex core polarity and the magnetization tilting induced by the IDMI boundary conditions. Micromagnetic simulations predict that a magnetoresistive memory based on the radial vortex state in both free and polarizer layers can be efficiently switched by a threshold current density smaller than 10^{6}  A/cm^{2}. The switching processes occur via the nucleation of topologically connected vortices and vortex-antivortex pairs, followed by spin-wave emissions due to vortex-antivortex annihilations. PMID:27588879

  16. Interactions between Trypanosoma cruzi Secreted Proteins and Host Cell Signaling Pathways

    PubMed Central

    Watanabe Costa, Renata; da Silveira, Jose F.; Bahia, Diana

    2016-01-01

    Chagas disease is one of the prevalent neglected tropical diseases, affecting at least 6–7 million individuals in Latin America. It is caused by the protozoan parasite Trypanosoma cruzi, which is transmitted to vertebrate hosts by blood-sucking insects. After infection, the parasite invades and multiplies in the myocardium, leading to acute myocarditis that kills around 5% of untreated individuals. T. cruzi secretes proteins that manipulate multiple host cell signaling pathways to promote host cell invasion. The primary secreted lysosomal peptidase in T. cruzi is cruzipain, which has been shown to modulate the host immune response. Cruzipain hinders macrophage activation during the early stages of infection by interrupting the NF-kB P65 mediated signaling pathway. This allows the parasite to survive and replicate, and may contribute to the spread of infection in acute Chagas disease. Another secreted protein P21, which is expressed in all of the developmental stages of T. cruzi, has been shown to modulate host phagocytosis signaling pathways. The parasite also secretes soluble factors that exert effects on host extracellular matrix, such as proteolytic degradation of collagens. Finally, secreted phospholipase A from T. cruzi contributes to lipid modifications on host cells and concomitantly activates the PKC signaling pathway. Here, we present a brief review of the interaction between secreted proteins from T. cruzi and the host cells, emphasizing the manipulation of host signaling pathways during invasion. PMID:27065960

  17. Characterization of the interactions between protein and carbon black.

    PubMed

    Chen, Tzu-Tao; Chuang, Kai-Jen; Chiang, Ling-Ling; Chen, Chun-Chao; Yeh, Chi-Tai; Wang, Liang-Shun; Gregory, Clive; Jones, Tim; BéruBé, Kelly; Lee, Chun-Nin; Chuang, Hsiao-Chi; Cheng, Tsun-Jen

    2014-01-15

    A considerable amount of studies have been conducted to investigate the interactions of biological fluids with nanoparticle surfaces, which exhibit a high affinity for proteins and particles. However, the mechanisms underlying these interactions have not been elucidated, particularly as they relate to human health. Using bovine serum albumin (BSA) and mice bronchoalveolar lavage fluid (BALF) as models for protein-particle conjugates, we characterized the physicochemical modifications of carbon blacks (CB) with 23nm or 65nm in diameter after protein treatment. Adsorbed BALF-containing proteins were quantified and identified by pathways, biological analyses and protein classification. Significant modifications of the physicochemistry of CB were induced by the addition of BSA. Enzyme modulators and hydrolase predominately interacted with CB, with protein-to-CB interactions that were associated with the coagulation pathways. Additionally, our results revealed that an acute-phase response could be activated by these proteins. With regard to human health, the present study revealed that the CB can react with proteins (∼55kDa and 70kDa) after inhalation and may modify the functional structures of lung proteins, leading to the activation of acute-inflammatory responses in the lungs. PMID:24291665

  18. Multiplex single-molecule interaction profiling of DNA barcoded proteins

    PubMed Central

    Gu, Liangcai; Li, Chao; Aach, John; Hill, David E.; Vidal, Marc; Church, George M.

    2014-01-01

    In contrast with advances in massively parallel DNA sequencing1, high-throughput protein analyses2-4 are often limited by ensemble measurements, individual analyte purification and hence compromised quality and cost-effectiveness. Single-molecule (SM) protein detection achieved using optical methods5 is limited by the number of spectrally nonoverlapping chromophores. Here, we introduce a single molecular interaction-sequencing (SMI-Seq) technology for parallel protein interaction profiling leveraging SM advantages. DNA barcodes are attached to proteins collectively via ribosome display6 or individually via enzymatic conjugation. Barcoded proteins are assayed en masse in aqueous solution and subsequently immobilized in a polyacrylamide (PAA) thin film to construct a random SM array, where barcoding DNAs are amplified into in situ polymerase colonies (polonies)7 and analyzed by DNA sequencing. This method allows precise quantification of various proteins with a theoretical maximum array density of over one million polonies per square millimeter. Furthermore, protein interactions can be measured based on the statistics of colocalized polonies arising from barcoding DNAs of interacting proteins. Two demanding applications, G-protein coupled receptor (GPCR) and antibody binding profiling, were demonstrated. SMI-Seq enables “library vs. library” screening in a one-pot assay, simultaneously interrogating molecular binding affinity and specificity. PMID:25252978

  19. Visualization and targeted disruption of protein interactions in living cells

    PubMed Central

    Herce, Henry D.; Deng, Wen; Helma, Jonas; Leonhardt, Heinrich; Cardoso, M. Cristina

    2013-01-01

    Protein–protein interactions are the basis of all processes in living cells, but most studies of these interactions rely on biochemical in vitro assays. Here we present a simple and versatile fluorescent-three-hybrid (F3H) strategy to visualize and target protein–protein interactions. A high-affinity nanobody anchors a GFP-fusion protein of interest at a defined cellular structure and the enrichment of red-labelled interacting proteins is measured at these sites. With this approach, we visualize the p53–HDM2 interaction in living cells and directly monitor the disruption of this interaction by Nutlin 3, a drug developed to boost p53 activity in cancer therapy. We further use this approach to develop a cell-permeable vector that releases a highly specific peptide disrupting the p53 and HDM2 interaction. The availability of multiple anchor sites and the simple optical readout of this nanobody-based capture assay enable systematic and versatile analyses of protein–protein interactions in practically any cell type and species. PMID:24154492

  20. Use of Flow Cytometric Methods to Quantify Protein-Protein Interactions

    PubMed Central

    Blazer, Levi L.; Roman, David L.; Muxlow, Molly R.; Neubig, Richard R.

    2010-01-01

    A method is described for the quantitative analysis of protein-protein interactions using the Flow Cytometry Protein Interaction Assay (FCPIA). This method is based upon immobilizing protein on a polystyrene bead, incubating these beads with a fluorescently labeled binding partner, and assessing the sample for bead-associated fluorescence in a flow cytometer. This method can be used to calculate protein-protein interaction affinities or to perform competition experiments with unlabeled binding partners or small molecules. Examples described in this protocol highlight the use of this assay in the quantification of the affinity of binding partners of the Regulator of G-Protein Signaling protein, RGS19, in either a saturation or competition format. An adaptation of this method that is compatible for High Throughput screening is also provided. PMID:20069525

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2016-01-01

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

  3. Nano-mechanical measurements of protein-DNA interactions with a silicon nitride pulley

    PubMed Central

    Shon, Min Ju; Cohen, Adam E.

    2016-01-01

    Proteins adhere to DNA at locations and with strengths that depend on the protein conformation, the underlying DNA sequence and the ionic content of the solution. A facile technique to probe the positions and strengths of protein-DNA binding would aid in understanding these important interactions. Here, we describe a ‘DNA pulley’ for position-resolved nano-mechanical measurements of protein-DNA interactions. A molecule of λ DNA is tethered by one end to a glass surface, and by the other end to a magnetic bead. The DNA is stretched horizontally by a magnet, and a nanoscale knife made of silicon nitride is manipulated to contact, bend and scan along the DNA. The mechanical profile of the DNA at the contact with the knife is probed via nanometer-precision optical tracking of the magnetic bead. This system enables detection of protein bumps on the DNA and localization of their binding sites. We study theoretically the technical requirements to detect mechanical heterogeneities in the DNA itself. PMID:26338777

  4. Nano-mechanical measurements of protein-DNA interactions with a silicon nitride pulley.

    PubMed

    Shon, Min Ju; Cohen, Adam E

    2016-01-01

    Proteins adhere to DNA at locations and with strengths that depend on the protein conformation, the underlying DNA sequence and the ionic content of the solution. A facile technique to probe the positions and strengths of protein-DNA binding would aid in understanding these important interactions. Here, we describe a 'DNA pulley' for position-resolved nano-mechanical measurements of protein-DNA interactions. A molecule of λ DNA is tethered by one end to a glass surface, and by the other end to a magnetic bead. The DNA is stretched horizontally by a magnet, and a nanoscale knife made of silicon nitride is manipulated to contact, bend and scan along the DNA. The mechanical profile of the DNA at the contact with the knife is probed via nanometer-precision optical tracking of the magnetic bead. This system enables detection of protein bumps on the DNA and localization of their binding sites. We study theoretically the technical requirements to detect mechanical heterogeneities in the DNA itself. PMID:26338777

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

    DOE PAGESBeta

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

    2006-01-01

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

  6. Assessing Energetic Contributions to Binding from a Disordered Region in a Protein-Protein Interaction

    SciTech Connect

    S Cho; C Swaminathan; D Bonsor; M Kerzic; R Guan; J Yang; C Kieke; P Anderson; D Kranz; et al.

    2011-12-31

    Many functional proteins are at least partially disordered prior to binding. Although the structural transitions upon binding of disordered protein regions can influence the affinity and specificity of protein complexes, their precise energetic contributions to binding are unknown. Here, we use a model protein-protein interaction system in which a locally disordered region has been modified by directed evolution to quantitatively assess the thermodynamic and structural contributions to binding of disorder-to-order transitions. Through X-ray structure determination of the protein binding partners before and after complex formation and isothermal titration calorimetry of the interactions, we observe a correlation between protein ordering and binding affinity for complexes along this affinity maturation pathway. Additionally, we show that discrepancies between observed and calculated heat capacities based on buried surface area changes in the protein complexes can be explained largely by heat capacity changes that would result solely from folding the locally disordered region. Previously developed algorithms for predicting binding energies of protein-protein interactions, however, are unable to correctly model the energetic contributions of the structural transitions in our model system. While this highlights the shortcomings of current computational methods in modeling conformational flexibility, it suggests that the experimental methods used here could provide training sets of molecular interactions for improving these algorithms and further rationalizing molecular recognition in protein-protein interactions.

  7. Analytical applications of partitioning in aqueous two-phase systems: Exploring protein structural changes and protein-partner interactions in vitro and in vivo by solvent interaction analysis method.

    PubMed

    Zaslavsky, Boris Y; Uversky, Vladimir N; Chait, Arnon

    2016-05-01

    This review covers the fundamentals of protein partitioning in aqueous two-phase systems (ATPS). Included is a review of advancements in the analytical application of solute partitioning in ATPS over the last two decades, with multiple examples of experimental data providing evidence that phase-forming polymers do not interact with solutes partitioned in ATPS. The partitioning of solutes is governed by the differences in solute interactions with aqueous media in the two phases. Solvent properties of the aqueous media in these two phases may be characterized and manipulated. The solvent interaction analysis (SIA) method, based on the solute partitioning in ATPS, may be used for characterization and analysis of individual proteins and their interactions with different partners. The current state of clinical proteomics regarding the discovery and monitoring of new protein biomarkers is discussed, and it is argued that the protein expression level in a biological fluid may be not the optimal focus of clinical proteomic research. Multiple examples of application of the SIA method for discovery of changes in protein structure and protein-partner interactions in biological fluids are described. The SIA method reveals new opportunities for discovery and monitoring structure-based protein biomarkers. PMID:26923390

  8. 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

  9. Mutual diffusion of interacting membrane proteins.

    PubMed Central

    Abney, J R; Scalettar, B A; Owicki, J C

    1989-01-01

    The generalized Stokes-Einstein equation is used, together with the two-dimensional pressure equation, to analyze mutual diffusion in concentrated membrane systems. These equations can be used to investigate the role that both direct and hydrodynamic interactions play in determining diffusive behavior. Here only direct interactions are explicitly incorporated into the theory at high densities; however, both direct and hydrodynamic interactions are analyzed for some dilute solutions. We look at diffusion in the presence of weak attractions, soft repulsions, and hard-core repulsions. It is found that, at low densities, attractions retard mutual diffusion while repulsions enhance it. Mechanistically, attractions tend to tether particles together and oppose the dissipation of gradients or fluctuations in concentration, while repulsions provide a driving force that pushes particles apart. At higher concentrations, changes in the structure of the fluid enhance mutual diffusion even in the presence of attractions. It is shown that the theoretical description of postelectrophoresis relaxation and fluorescence correlation spectroscopy experiments must be modified if interacting systems are studied. The effects of interactions on mutual diffusion coefficients have probably already been seen in postelectrophoresis relaxation experiments. PMID:2775829

  10. Investigation of protein-ligand interactions by mass spectrometry.

    PubMed

    Sinz, Andrea

    2007-04-01

    The rate of drug discovery is greatly dependent on the development and improvement of rapid and reliable analytical methods that allow screening for protein-ligand interactions. The solution-based methods for investigating protein-ligand interactions by mass spectrometry (MS), which are discussed in this paper, are hydrogen/deuterium exchange of protein backbone amide hydrogens, and photoaffinity labeling. Moreover, MS analysis of intact noncovalent protein-ligand complexes is described. Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS) with its ultra-high resolution and excellent mass accuracy is also considered herein as it is gaining increasing popularity for a mass spectrometric investigation of protein-ligand interactions. PMID:17299828

  11. Neurodegenerative diseases: quantitative predictions of protein-RNA interactions.

    PubMed

    Cirillo, Davide; Agostini, Federico; Klus, Petr; Marchese, Domenica; Rodriguez, Silvia; Bolognesi, Benedetta; Tartaglia, Gian Gaetano

    2013-02-01

    Increasing evidence indicates that RNA plays an active role in a number of neurodegenerative diseases. We recently introduced a theoretical framework, catRAPID, to predict the binding ability of protein and RNA molecules. Here, we use catRAPID to investigate ribonucleoprotein interactions linked to inherited intellectual disability, amyotrophic lateral sclerosis, Creutzfeuld-Jakob, Alzheimer's, and Parkinson's diseases. We specifically focus on (1) RNA interactions with fragile X mental retardation protein FMRP; (2) protein sequestration caused by CGG repeats; (3) noncoding transcripts regulated by TAR DNA-binding protein 43 TDP-43; (4) autogenous regulation of TDP-43 and FMRP; (5) iron-mediated expression of amyloid precursor protein APP and α-synuclein; (6) interactions between prions and RNA aptamers. Our results are in striking agreement with experimental evidence and provide new insights in processes associated with neuronal function and misfunction. PMID:23264567

  12. 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

  13. 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

  14. Membrane interaction of retroviral Gag proteins

    PubMed Central

    Dick, Robert A.; Vogt, Volker M.

    2014-01-01

    Assembly of an infectious retroviral particle relies on multimerization of the Gag polyprotein at the inner leaflet of the plasma membrane. The three domains of Gag common to all retroviruses – MA, CA, and NC – provide the signals for membrane binding, assembly, and viral RNA packaging, respectively. These signals do not function independently of one another. For example, Gag multimerization enhances membrane binding and is more efficient when NC is interacting with RNA. MA binding to the plasma membrane is governed by several principles, including electrostatics, recognition of specific lipid head groups, hydrophobic interactions, and membrane order. HIV-1 uses many of these principles while Rous sarcoma virus (RSV) appears to use fewer. This review describes the principles that govern Gag interactions with membranes, focusing on RSV and HIV-1 Gag. The review also defines lipid and membrane behavior, and discusses the complexities in determining how lipid and membrane behavior impact Gag membrane binding. PMID:24808894

  15. A protein interaction map for cell polarity development.

    PubMed

    Drees, B L; Sundin, B; Brazeau, E; Caviston, J P; Chen, G C; Guo, W; Kozminski, K G; Lau, M W; Moskow, J J; Tong, A; Schenkman, L R; McKenzie, A; Brennwald, P; Longtine, M; Bi, E; Chan, C; Novick, P; Boone, C; Pringle, J R; Davis, T N; Fields, S; Drubin, D G

    2001-08-01

    Many genes required for cell polarity development in budding yeast have been identified and arranged into a functional hierarchy. Core elements of the hierarchy are widely conserved, underlying cell polarity development in diverse eukaryotes. To enumerate more fully the protein-protein interactions that mediate cell polarity development, and to uncover novel mechanisms that coordinate the numerous events involved, we carried out a large-scale two-hybrid experiment. 68 Gal4 DNA binding domain fusions of yeast proteins associated with the actin cytoskeleton, septins, the secretory apparatus, and Rho-type GTPases were used to screen an array of yeast transformants that express approximately 90% of the predicted Saccharomyces cerevisiae open reading frames as Gal4 activation domain fusions. 191 protein-protein interactions were detected, of which 128 had not been described previously. 44 interactions implicated 20 previously uncharacterized proteins in cell polarity development. Further insights into possible roles of 13 of these proteins were revealed by their multiple two-hybrid interactions and by subcellular localization. Included in the interaction network were associations of Cdc42 and Rho1 pathways with proteins involved in exocytosis, septin organization, actin assembly, microtubule organization, autophagy, cytokinesis, and cell wall synthesis. Other interactions suggested direct connections between Rho1- and Cdc42-regulated pathways; the secretory apparatus and regulators of polarity establishment; actin assembly and the morphogenesis checkpoint; and the exocytic and endocytic machinery. In total, a network of interactions that provide an integrated response of signaling proteins, the cytoskeleton, and organelles to the spatial cues that direct polarity development was revealed. PMID:11489916

  16. PIPs: human protein–protein interaction prediction database

    PubMed Central

    McDowall, Mark D.; Scott, Michelle S.; Barton, Geoffrey J.

    2009-01-01

    The PIPs database (http://www.compbio.dundee.ac.uk/www-pips) is a resource for studying protein–protein interactions in human. It contains predictions of >37 000 high probability interactions of which >34 000 are not reported in the interaction databases HPRD, BIND, DIP or OPHID. The interactions in PIPs were calculated by a Bayesian method that combines information from expression, orthology, domain co-occurrence, post-translational modifications and sub-cellular location. The predictions also take account of the topology of the predicted interaction network. The web interface to PIPs ranks predictions according to their likelihood of interaction broken down by the contribution from each information source and with easy access to the evidence that supports each prediction. Where data exists in OPHID, HPRD, DIP or BIND for a protein pair this is also reported in the output tables returned by a search. A network browser is included to allow convenient browsing of the interaction network for any protein in the database. The PIPs database provides a new resource on protein–protein interactions in human that is straightforward to browse, or can be exploited completely, for interaction network modelling. PMID:18988626

  17. Biophysics of protein-DNA interactions and chromosome organization

    PubMed Central

    Marko, John F.

    2014-01-01

    The function of DNA in cells depends on its interactions with protein molecules, which recognize and act on base sequence patterns along the double helix. These notes aim to introduce basic polymer physics of DNA molecules, biophysics of protein-DNA interactions and their study in single-DNA experiments, and some aspects of large-scale chromosome structure. Mechanisms for control of chromosome topology will also be discussed. PMID:25419039

  18. Overview of the protein-protein interaction annotation extraction task of BioCreative II

    PubMed Central

    Krallinger, Martin; Leitner, Florian; Rodriguez-Penagos, Carlos; Valencia, Alfonso

    2008-01-01

    Background: The biomedical literature is the primary information source for manual protein-protein interaction annotations. Text-mining systems have been implemented to extract binary protein interactions from articles, but a comprehensive comparison between the different techniques as well as with manual curation was missing. Results: We designed a community challenge, the BioCreative II protein-protein interaction (PPI) task, based on the main steps of a manual protein interaction annotation workflow. It was structured into four distinct subtasks related to: (a) detection of protein interaction-relevant articles; (b) extraction and normalization of protein interaction pairs; (c) retrieval of the interaction detection methods used; and (d) retrieval of actual text passages that provide evidence for protein interactions. A total of 26 teams submitted runs for at least one of the proposed subtasks. In the interaction article detection subtask, the top scoring team reached an F-score of 0.78. In the interaction pair extraction and mapping to SwissProt, a precision of 0.37 (with recall of 0.33) was obtained. For associating articles with an experimental interaction detection method, an F-score of 0.65 was achieved. As for the retrieval of the PPI passages best summarizing a given protein interaction in full-text articles, 19% of the submissions returned by one of the runs corresponded to curator-selected sentences. Curators extracted only the passages that best summarized a given interaction, implying that many of the automatically extracted ones could contain interaction information but did not correspond to the most informative sentences. Conclusion: The BioCreative II PPI task is the first attempt to compare the performance of text-mining tools specific for each of the basic steps of the PPI extraction pipeline. The challenges identified range from problems in full-text format conversion of articles to difficulties in detecting interactor protein pairs and then

  19. The Intraviral Protein Interaction Network of Hepatitis C Virus*

    PubMed Central

    Hagen, Nicole; Bayer, Karen; Rösch, Kathrin; Schindler, Michael

    2014-01-01

    Hepatitis C virus (HCV) is a global health problem and one of the main reasons for chronic liver diseases such as cirrhosis and hepatocellular carcinoma. The HCV genome is translated into a polyprotein which is proteolytically processed into 10 viral proteins. The interactome of the HCV proteins with the host cell has been worked out; however, it remains unclear how viral proteins interact with each other. We aimed to generate the interaction network of these 10 HCV proteins using a flow-cytometry-based FRET assay established in our laboratory (Banning, C., Votteler, J., Hoffmann, D., Koppensteiner, H., Warmer, M., Reimer, R., Kirchhoff, F., Schubert, U., Hauber, J., and Schindler, M. (2010) A flow cytometry-based FRET assay to identify and analyse protein-protein interactions in living cells. PLoS One 5, e9344). HCV proteins were constructed as fusions with the chromophores CFP and YFP. All HCV fusions were expressed and localized to specific subcellular compartments, indicating that they were functional. FACS-FRET measurements identified a total of 20 interactions; 13 of these were previously described and have now been confirmed in living cells via our method. Among the seven novel protein binding pairs, HCV p7 plays a pivotal role. It binds to the HCV capsid protein Core and the two glycoproteins E1 and E2. These interplays were further demonstrated in the relevant context of Huh7.5 liver cells expressing infectious HCV. Our work demonstrates the feasibility of rapidly generating small interaction networks via FACS-FRET and defines the network of intra-HCV protein interactions. Furthermore, our data support an important role of p7 in HCV assembly. PMID:24797426

  20. Linkers in the structural biology of protein–protein interactions

    PubMed Central

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

    2013-01-01

    Linkers or spacers are short amino acid sequences created in nature to separate multiple domains in a single protein. Most of them are rigid and function to prohibit unwanted interactions between the discrete domains. However, Gly-rich linkers are flexible, connecting various domains in a single protein without interfering with the function of each domain. The advent of recombinant DNA technology made it possible to fuse two interacting partners with the introduction of artificial linkers. Often, independent proteins may not exist as stable or structured proteins until they interact with their binding partner, following which they gain stability and the essential structural elements. Gly-rich linkers have been proven useful for these types of unstable interactions, particularly where the interaction is weak and transient, by creating a covalent link between the proteins to form a stable protein–protein complex. Gly-rich linkers are also employed to form stable covalently linked dimers, and to connect two independent domains that create a ligand-binding site or recognition sequence. The lengths of linkers vary from 2 to 31 amino acids, optimized for each condition so that the linker does not impose any constraints on the conformation or interactions of the linked partners. Various structures of covalently linked protein complexes have been described using X-ray crystallography, nuclear magnetic resonance and cryo-electron microscopy techniques. In this review, we evaluate several structural studies where linkers have been used to improve protein quality, to produce stable protein–protein complexes, and to obtain protein dimers. PMID:23225024

  1. Sequence Motifs in MADS Transcription Factors Responsible for Specificity and Diversification of Protein-Protein Interaction

    PubMed Central

    van Dijk, Aalt D. J.; Morabito, Giuseppa; Fiers, Martijn; van Ham, Roeland C. H. J.; Angenent, Gerco C.; Immink, Richard G. H.

    2010-01-01

    Protein sequences encompass tertiary structures and contain information about specific molecular interactions, which in turn determine biological functions of proteins. Knowledge about how protein sequences define interaction specificity is largely missing, in particular for paralogous protein families with high sequence similarity, such as the plant MADS domain transcription factor family. In comparison to the situation in mammalian species, this important family of transcription regulators has expanded enormously in plant species and contains over 100 members in the model plant species Arabidopsis thaliana. Here, we provide insight into the mechanisms that determine protein-protein interaction specificity for the Arabidopsis MADS domain transcription factor family, using an integrated computational and experimental approach. Plant MADS proteins have highly similar amino acid sequences, but their dimerization patterns vary substantially. Our computational analysis uncovered small sequence regions that explain observed differences in dimerization patterns with reasonable accuracy. Furthermore, we show the usefulness of the method for prediction of MADS domain transcription factor interaction networks in other plant species. Introduction of mutations in the predicted interaction motifs demonstrated that single amino acid mutations can have a large effect and lead to loss or gain of specific interactions. In addition, various performed bioinformatics analyses shed light on the way evolution has shaped MADS domain transcription factor interaction specificity. Identified protein-protein interaction motifs appeared to be strongly conserved among orthologs, indicating their evolutionary importance. We also provide evidence that mutations in these motifs can be a source for sub- or neo-functionalization. The analyses presented here take us a step forward in understanding protein-protein interactions and the interplay between protein sequences and network evolution. PMID

  2. A conserved patch of hydrophobic amino acids modulates Myb activity by mediating protein-protein interactions.

    PubMed

    Dukare, Sandeep; Klempnauer, Karl-Heinz

    2016-07-01

    The transcription factor c-Myb plays a key role in the control of proliferation and differentiation in hematopoietic progenitor cells and has been implicated in the development of leukemia and certain non-hematopoietic tumors. c-Myb activity is highly dependent on the interaction with the coactivator p300 which is mediated by the transactivation domain of c-Myb and the KIX domain of p300. We have previously observed that conservative valine-to-isoleucine amino acid substitutions in a conserved stretch of hydrophobic amino acids have a profound effect on Myb activity. Here, we have explored the function of the hydrophobic region as a mediator of protein-protein interactions. We show that the hydrophobic region facilitates Myb self-interaction and binding of the histone acetyl transferase Tip60, a previously identified Myb interacting protein. We show that these interactions are affected by the valine-to-isoleucine amino acid substitutions and suppress Myb activity by interfering with the interaction of Myb and the KIX domain of p300. Taken together, our work identifies the hydrophobic region in the Myb transactivation domain as a binding site for homo- and heteromeric protein interactions and leads to a picture of the c-Myb transactivation domain as a composite protein binding region that facilitates interdependent protein-protein interactions of Myb with regulatory proteins. PMID:27080133

  3. Screening for in planta protein-protein interactions combining bimolecular fluorescence complementation with flow cytometry

    PubMed Central

    2012-01-01

    Understanding protein and gene function requires identifying interaction partners using biochemical, molecular or genetic tools. In plants, searching for novel protein-protein interactions is limited to protein purification assays, heterologous in vivo systems such as the yeast-two-hybrid or mutant screens. Ideally one would be able to search for novel protein partners in living plant cells. We demonstrate that it is possible to screen for novel protein-protein interactions from a random library in protoplasted Arabidopsis plant cells and recover some of the interacting partners. Our screen is based on capturing the bi-molecular complementation of mYFP between an YN-bait fusion partner and a completely random prey YC-cDNA library with FACS. The candidate interactions were confirmed using in planta BiFC assays and in planta FRET-FLIM assays. From this work, we show that the well characterized protein Calcium Dependent Protein Kinase 3 (CPK3) interacts with APX3, HMGB5, ORP2A and a ricin B-related lectin domain containing protein At2g39050. This is one of the first randomin planta screens to be successfully employed. PMID:22789293

  4. Discovering Distinct Functional Modules of Specific Cancer Types Using Protein-Protein Interaction Networks

    PubMed Central

    Shen, Ru; Wang, Xiaosheng; Guda, Chittibabu

    2015-01-01

    Background. The molecular profiles exhibited in different cancer types are very different; hence, discovering distinct functional modules associated with specific cancer types is very important to understand the distinct functions associated with them. Protein-protein interaction networks carry vital information about molecular interactions in cellular systems, and identification of functional modules (subgraphs) in these networks is one of the most important applications of biological network analysis. Results. In this study, we developed a new graph theory based method to identify distinct functional modules from nine different cancer protein-protein interaction networks. The method is composed of three major steps: (i) extracting modules from protein-protein interaction networks using network clustering algorithms; (ii) identifying distinct subgraphs from the derived modules; and (iii) identifying distinct subgraph patterns from distinct subgraphs. The subgraph patterns were evaluated using experimentally determined cancer-specific protein-protein interaction data from the Ingenuity knowledgebase, to identify distinct functional modules that are specific to each cancer type. Conclusion. We identified cancer-type specific subgraph patterns that may represent the functional modules involved in the molecular pathogenesis of different cancer types. Our method can serve as an effective tool to discover cancer-type specific functional modules from large protein-protein interaction networks. PMID:26495282

  5. Identification of Protein Interactions Involved in Cellular Signaling

    PubMed Central

    Westermarck, Jukka; Ivaska, Johanna; Corthals, Garry L.

    2013-01-01

    Protein-protein interactions drive biological processes. They are critical for all intra- and extracellular functions, and the technologies to analyze them are widely applied throughout the various fields of biological sciences. This study takes an in-depth view of some common principles of cellular regulation and provides a detailed account of approaches required to comprehensively map signaling protein-protein interactions in any particular cellular system or condition. We provide a critical review of the benefits and disadvantages of the yeast two-hybrid method and affinity purification coupled with mass spectrometric procedures for identification of signaling protein-protein interactions. In particular, we emphasize the quantitative and qualitative differences between tandem affinity and one-step purification (such as FLAG and Strep tag) methods. Although applicable to all types of interaction studies, a special section is devoted in this review to aspects that should be considered when attempting to identify signaling protein interactions that often are transient and weak by nature. Finally, we discuss shotgun and quantitative information that can be gleaned by MS-coupled methods for analysis of multiprotein complexes. PMID:23481661

  6. Quantification of protein interaction kinetics in a micro droplet

    NASA Astrophysics Data System (ADS)

    Yin, L. L.; Wang, S. P.; Shan, X. N.; Zhang, S. T.; Tao, N. J.

    2015-11-01

    Characterization of protein interactions is essential to the discovery of disease biomarkers, the development of diagnostic assays, and the screening for therapeutic drugs. Conventional flow-through kinetic measurements need relative large amount of sample that is not feasible for precious protein samples. We report a novel method to measure protein interaction kinetics in a single droplet with sub microliter or less volume. A droplet in a humidity-controlled environmental chamber is replacing the microfluidic channels as the reactor for the protein interaction. The binding process is monitored by a surface plasmon resonance imaging (SPRi) system. Association curves are obtained from the average SPR image intensity in the center area of the droplet. The washing step required by conventional flow-through SPR method is eliminated in the droplet method. The association and dissociation rate constants and binding affinity of an antigen-antibody interaction are obtained by global fitting of association curves at different concentrations. The result obtained by this method is accurate as validated by conventional flow-through SPR system. This droplet-based method not only allows kinetic studies for proteins with limited supply but also opens the door for high-throughput protein interaction study in a droplet-based microarray format that enables measurement of many to many interactions on a single chip.

  7. PLIP: fully automated protein-ligand interaction profiler.

    PubMed

    Salentin, Sebastian; Schreiber, Sven; Haupt, V Joachim; Adasme, Melissa F; Schroeder, Michael

    2015-07-01

    The characterization of interactions in protein-ligand complexes is essential for research in structural bioinformatics, drug discovery and biology. However, comprehensive tools are not freely available to the research community. Here, we present the protein-ligand interaction profiler (PLIP), a novel web service for fully automated detection and visualization of relevant non-covalent protein-ligand contacts in 3D structures, freely available at projects.biotec.tu-dresden.de/plip-web. The input is either a Protein Data Bank structure, a protein or ligand name, or a custom protein-ligand complex (e.g. from docking). In contrast to other tools, the rule-based PLIP algorithm does not require any structure preparation. It returns a list of detected interactions on single atom level, covering seven interaction types (hydrogen bonds, hydrophobic contacts, pi-stacking, pi-cation interactions, salt bridges, water bridges and halogen bonds). PLIP stands out by offering publication-ready images, PyMOL session files to generate custom images and parsable result files to facilitate successive data processing. The full python source code is available for download on the website. PLIP's command-line mode allows for high-throughput interaction profiling. PMID:25873628

  8. Quantification of protein interaction kinetics in a micro droplet

    SciTech Connect

    Yin, L. L.; Wang, S. P. E-mail: njtao@asu.edu; Shan, X. N.; Tao, N. J. E-mail: njtao@asu.edu; Zhang, S. T.

    2015-11-15

    Characterization of protein interactions is essential to the discovery of disease biomarkers, the development of diagnostic assays, and the screening for therapeutic drugs. Conventional flow-through kinetic measurements need relative large amount of sample that is not feasible for precious protein samples. We report a novel method to measure protein interaction kinetics in a single droplet with sub microliter or less volume. A droplet in a humidity-controlled environmental chamber is replacing the microfluidic channels as the reactor for the protein interaction. The binding process is monitored by a surface plasmon resonance imaging (SPRi) system. Association curves are obtained from the average SPR image intensity in the center area of the droplet. The washing step required by conventional flow-through SPR method is eliminated in the droplet method. The association and dissociation rate constants and binding affinity of an antigen-antibody interaction are obtained by global fitting of association curves at different concentrations. The result obtained by this method is accurate as validated by conventional flow-through SPR system. This droplet-based method not only allows kinetic studies for proteins with limited supply but also opens the door for high-throughput protein interaction study in a droplet-based microarray format that enables measurement of many to many interactions on a single chip.

  9. Ex vivo identification of protein-protein interactions involving the dopamine transporter.

    PubMed

    Hadlock, Gregory C; Nelson, Chad C; Baucum, Anthony J; Hanson, Glen R; Fleckenstein, Annette E

    2011-03-30

    The dopamine (DA) transporter (DAT) is a key regulator of dopaminergic signaling as it mediates the reuptake of extrasynaptic DA and thereby terminates dopaminergic signaling. Emerging evidence indicates that DAT function is influenced through interactions with other proteins. The current report describes a method to identify such interactions following DAT immunoprecipitation from a rat striatal synaptosomal preparation. This subcellular fraction was selected since DAT function is often determined ex vivo by measuring DA uptake in this preparation and few reports investigating DAT-protein interactions have utilized this preparation. Following SDS-PAGE and colloidal Coomassie staining, selected protein bands from a DAT-immunoprecipitate were excised, digested with trypsin, extracted, and analyzed by liquid chromatography tandem mass spectrometry (LC/MS/MS). From the analysis of the tryptic peptides, several proteins were identified including DAT, Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) β, CaMKII δ, protein kinase C (PKC) β, and PKC γ. Co-immunoprecipitation of PKC, CaMKII, and protein interacting with C kinase-1 with DAT was confirmed by Western blotting. Thus, the present study highlights a method to immunoprecipitate DAT and to identify co-immunoprecipitating proteins using LC/MS/MS and Western blotting. This method can be utilized to evaluate DAT protein-protein interactions but also to assess interactions involving other synaptic proteins. Ex vivo identification of protein-protein interactions will provide new insight into the function and regulation of a variety of synaptic, membrane-associated proteins, including DAT. PMID:21291912

  10. Gel mobility shift assays to detect protein-RNA interactions.

    PubMed

    Yakhnin, Alexander V; Yakhnin, Helen; Babitzke, Paul

    2012-01-01

    The gel mobility shift assay is a powerful technique for detecting and quantifying protein-RNA interactions. While other techniques such as filter binding and isothermal titration calorimetry (ITC) are available for quantifying protein-RNA interactions, gel shift analysis provides the added advantage that you can visualize the protein-RNA complexes. In the gel shift assay, protein-RNA complexes are typically separated from the unbound RNA using native polyacrylamide gels in Tris/borate/EDTA buffer, although an alternative Tris-glycine buffering system is superior in many situations. Here, we describe both gel shift methods, along with strategies to improve separation of protein-RNA complexes from free RNA, which can be a particular challenge for small RNA binding proteins. PMID:22736005

  11. Proteins differentially interact with grapefruit furanocoumarins

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Grapefruit juice (GFJ) interferes with the cytochrome P450 3A4 activity responsible for metabolizing certain medications. This interference is referred to as the "grapefruit-drug interaction". Grapefruit furanocoumarins (FCs), such as 6', 7'-dihydroxybergamottin (DHB) and bergamottin (BM), have been...

  12. cAMP-dependent protein kinase and lipolysis in rat adipocytes. I. Cell preparation, manipulation, and predictability in behavior.

    PubMed

    Honnor, R C; Dhillon, G S; Londos, C

    1985-12-01

    With the use of -cAMP/+cAMP activity ratios of cAMP-dependent protein kinase (A-kinase) in fat cell extracts as an index of cellular cAMP concentrations, it is apparent from both the current literature and from data presented in this paper that classical cell isolation procedures yield cells whose behavior is unpredictable from day to day. Herein, procedures are described for isolating adipocytes, preparing cytosolic extracts, and assaying A-kinase that result in kinase activity ratios in isolated cells equal to those in the fat pad from which cells are derived, approximately 0.05. An important modification in the procedure is the inclusion of 200 nM exogenous Ado in all cell manipulation media, and the data indicate that variable removal of contaminating endogenous Ado accounts for unpredictable results with standard cell isolation techniques. A further benefit of Ado inclusion is greatly reduced cell lysis. Acute removal of Ado with adenosine deaminase results in rapid elevation of A-kinase activity ratios and lipolysis which, in fasted animals, equals that achieved with lipolytic hormones. Cells from fed animals exhibit poor predictability in behavior. Moreover, A-kinase activity ratios exhibit seasonal tendencies in response to Ado removal, with cells isolated in spring being more activated than cells isolated later in the year. The information and procedures in this paper form the basis for succeeding papers on the regulation of adipocyte metabolism by hormones. PMID:2415513

  13. 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

  14. Computational Prediction of Protein–Protein Interaction Networks: Algo-rithms and Resources

    PubMed Central

    Zahiri, Javad; Bozorgmehr, Joseph Hannon; Masoudi-Nejad, Ali

    2013-01-01

    Protein interactions play an important role in the discovery of protein functions and pathways in biological processes. This is especially true in case of the diseases caused by the loss of specific protein-protein interactions in the organism. The accuracy of experimental results in finding protein-protein interactions, however, is rather dubious and high throughput experimental results have shown both high false positive beside false negative information for protein interaction. Computational methods have attracted tremendous attention among biologists because of the ability to predict protein-protein interactions and validate the obtained experimental results. In this study, we have reviewed several computational methods for protein-protein interaction prediction as well as describing major databases, which store both predicted and detected protein-protein interactions, and the tools used for analyzing protein interaction networks and improving protein-protein interaction reliability. PMID:24396273

  15. Analysis of Lipolytic Protein Trafficking and Interactions in Adipocytes

    Technology Transfer Automated Retrieval System (TEKTRAN)

    This work examined the colocalization, trafficking, and interactions of key proteins involved in lipolysis during brief cAMP-dependent protein kinase A (PKA) activation. Double label immunofluorescence analysis of 3T3-L1 adipocytes indicated that PKA activation increases the translocation of hormon...

  16. Understanding Protein Synthesis: An Interactive Card Game Discussion

    ERIC Educational Resources Information Center

    Lewis, Alison; Peat, Mary; Franklin, Sue

    2005-01-01

    Protein synthesis is a complex process and students find it difficult to understand. This article describes an interactive discussion "game" used by first year biology students at the University of Sydney. The students, in small groups, use the game in which the processes of protein synthesis are actioned by the students during a practical…

  17. The Role of Protein-Protein and Protein-Membrane Interactions on P450 Function.

    PubMed

    Scott, Emily E; Wolf, C Roland; Otyepka, Michal; Humphreys, Sara C; Reed, James R; Henderson, Colin J; McLaughlin, Lesley A; Paloncýová, Markéta; Navrátilová, Veronika; Berka, Karel; Anzenbacher, Pavel; Dahal, Upendra P; Barnaba, Carlo; Brozik, James A; Jones, Jeffrey P; Estrada, D Fernando; Laurence, Jennifer S; Park, Ji Won; Backes, Wayne L

    2016-04-01

    This symposium summary, sponsored by the ASPET, was held at Experimental Biology 2015 on March 29, 2015, in Boston, Massachusetts. The symposium focused on: 1) the interactions of cytochrome P450s (P450s) with their redox partners; and 2) the role of the lipid membrane in their orientation and stabilization. Two presentations discussed the interactions of P450s with NADPH-P450 reductase (CPR) and cytochrome b5. First, solution nuclear magnetic resonance was used to compare the protein interactions that facilitated either the hydroxylase or lyase activities of CYP17A1. The lyase interaction was stimulated by the presence of b5 and 17α-hydroxypregnenolone, whereas the hydroxylase reaction was predominant in the absence of b5. The role of b5 was also shown in vivo by selective hepatic knockout of b5 from mice expressing CYP3A4 and CYP2D6; the lack of b5 caused a decrease in the clearance of several substrates. The role of the membrane on P450 orientation was examined using computational methods, showing that the proximal region of the P450 molecule faced the aqueous phase. The distal region, containing the substrate-access channel, was associated with the membrane. The interaction of NADPH-P450 reductase (CPR) with the membrane was also described, showing the ability of CPR to "helicopter" above the membrane. Finally, the endoplasmic reticulum (ER) was shown to be heterogeneous, having ordered membrane regions containing cholesterol and more disordered regions. Interestingly, two closely related P450s, CYP1A1 and CYP1A2, resided in different regions of the ER. The structural characteristics of their localization were examined. These studies emphasize the importance of P450 protein organization to their function. PMID:26851242

  18. Exosome engineering for efficient intracellular delivery of soluble proteins using optically reversible protein-protein interaction module.

    PubMed

    Yim, Nambin; Ryu, Seung-Wook; Choi, Kyungsun; Lee, Kwang Ryeol; Lee, Seunghee; Choi, Hojun; Kim, Jeongjin; Shaker, Mohammed R; Sun, Woong; Park, Ji-Ho; Kim, Daesoo; Heo, Won Do; Choi, Chulhee

    2016-01-01

    Nanoparticle-mediated delivery of functional macromolecules is a promising method for treating a variety of human diseases. Among nanoparticles, cell-derived exosomes have recently been highlighted as a new therapeutic strategy for the in vivo delivery of nucleotides and chemical drugs. Here we describe a new tool for intracellular delivery of target proteins, named 'exosomes for protein loading via optically reversible protein-protein interactions' (EXPLORs). By integrating a reversible protein-protein interaction module controlled by blue light with the endogenous process of exosome biogenesis, we are able to successfully load cargo proteins into newly generated exosomes. Treatment with protein-loaded EXPLORs is shown to significantly increase intracellular levels of cargo proteins and their function in recipient cells in vitro and in vivo. These results clearly indicate the potential of EXPLORs as a mechanism for the efficient intracellular transfer of protein-based therapeutics into recipient cells and tissues. PMID:27447450

  19. Identification of Redox and Glucose-Dependent Txnip Protein Interactions

    PubMed Central

    Neuharth, Skyla; Kim, Dae In; Motamedchaboki, Khatereh; Roux, Kyle J.

    2016-01-01

    Thioredoxin-interacting protein (Txnip) acts as a negative regulator of thioredoxin function and is a critical modulator of several diseases including, but not limited to, diabetes, ischemia-reperfusion cardiac injury, and carcinogenesis. Therefore, Txnip has become an attractive therapeutic target to alleviate disease pathologies. Although Txnip has been implicated with numerous cellular processes such as proliferation, fatty acid and glucose metabolism, inflammation, and apoptosis, the molecular mechanisms underlying these processes are largely unknown. The objective of these studies was to identify Txnip interacting proteins using the proximity-based labeling method, BioID, to understand differential regulation of pleiotropic Txnip cellular functions. The BioID transgene fused to Txnip expressed in HEK293 identified 31 interacting proteins. Many protein interactions were redox-dependent and were disrupted through mutation of a previously described reactive cysteine (C247S). Furthermore, we demonstrate that this model can be used to identify dynamic Txnip interactions due to known physiological regulators such as hyperglycemia. These data identify novel Txnip protein interactions and demonstrate dynamic interactions dependent on redox and glucose perturbations, providing clarification to the pleiotropic cellular functions of Txnip. PMID:27437069

  20. Examination of Interactions of Oppositely Charged Proteins in Gels

    SciTech Connect

    Ramasamy,P.; El-Maghrabi, M.; Halada, G.; Miller, L.; Rafailovich, M.

    2007-01-01

    Understanding the interactions of proteins with one another serves as an important step for developing faster protein separation methods. To examine protein-protein interactions of oppositely charged proteins, fluorescently labeled albumin and poly-L-lysine were subjected to electrophoresis in agarose gels, in which the cationic albumin and the anionic poly-L-lysine were allowed to migrate toward each other and interact. Fluorescence microscopy was used to image fluorescently tagged proteins in the gel. The secondary structure of the proteins in solution was studied using conventional FTIR spectroscopy. Results showed that sharp interfaces were formed where FITC tagged albumin met poly-L-lysine and that the interfaces did not migrate after they had been formed. The position of the interface in the gel was found to be linearly dependent upon the relative concentration of the proteins. The formation of the interface also depended upon the fluorescent tag attached to the protein. The size of the aggregates at the interface, the fluorescence intensity modifications, and the mobility of the interface for different pore sizes of the gel were investigated. It was observed that the interface was made up of aggregates of about 1 {mu}m in size. Using dynamic light scattering, it was observed that the size of the aggregates that formed due to interactions of oppositely charged proteins depended upon the fluorescent tags attached to the proteins. The addition of small amounts of poly-L-lysine to solutions containing FITC albumin decreased the zeta potential drastically. For this, we propose a model suggesting that adding small amounts of poly-L-lysine to solutions containing FITC -albumin favors the formation of macromolecular complexes having FITC albumin molecules on its surface. Although oppositely charged FITC tagged poly-L-lysine and FITC tagged albumin influence each other's migration velocities by forming aggregates, there were no observable secondary structural

  1. Genome-wide Protein-protein Interaction Screening by Protein-fragment Complementation Assay (PCA) in Living Cells

    PubMed Central

    Filteau, Marie; Leducq, Jean-Baptiste; Dubé, Alexandre K.; Landry, Christian R.

    2015-01-01

    Proteins are the building blocks, effectors and signal mediators of cellular processes. A protein’s function, regulation and localization often depend on its interactions with other proteins. Here, we describe a protocol for the yeast protein-fragment complementation assay (PCA), a powerful method to detect direct and proximal associations between proteins in living cells. The interaction between two proteins, each fused to a dihydrofolate reductase (DHFR) protein fragment, translates into growth of yeast strains in presence of the drug methotrexate (MTX). Differential fitness, resulting from different amounts of reconstituted DHFR enzyme, can be quantified on high-density colony arrays, allowing to differentiate interacting from non-interacting bait-prey pairs. The high-throughput protocol presented here is performed using a robotic platform that parallelizes mating of bait and prey strains carrying complementary DHFR-fragment fusion proteins and the survival assay on MTX. This protocol allows to systematically test for thousands of protein-protein interactions (PPIs) involving bait proteins of interest and offers several advantages over other PPI detection assays, including the study of proteins expressed from their endogenous promoters without the need for modifying protein localization and for the assembly of complex reporter constructs. PMID:25867901

  2. Molecular tweezers modulate 14-3-3 protein-protein interactions.

    PubMed

    Bier, David; Rose, Rolf; Bravo-Rodriguez, Kenny; Bartel, Maria; Ramirez-Anguita, Juan Manuel; Dutt, Som; Wilch, Constanze; Klärner, Frank-Gerrit; Sanchez-Garcia, Elsa; Schrader, Thomas; Ottmann, Christian

    2013-03-01

    Supramolecular chemistry has recently emerged as a promising way to modulate protein functions, but devising molecules that will interact with a protein in the desired manner is difficult as many competing interactions exist in a biological environment (with solvents, salts or different sites for the target biomolecule). We now show that lysine-specific molecular tweezers bind to a 14-3-3 adapter protein and modulate its interaction with partner proteins. The tweezers inhibit binding between the 14-3-3 protein and two partner proteins--a phosphorylated (C-Raf) protein and an unphosphorylated one (ExoS)--in a concentration-dependent manner. Protein crystallography shows that this effect arises from the binding of the tweezers to a single surface-exposed lysine (Lys214) of the 14-3-3 protein in the proximity of its central channel, which normally binds the partner proteins. A combination of structural analysis and computer simulations provides rules for the tweezers' binding preferences, thus allowing us to predict their influence on this type of protein-protein interactions. PMID:23422566

  3. Lipid-protein interactions: Lessons learned from stress.

    PubMed

    Battle, A R; Ridone, P; Bavi, N; Nakayama, Y; Nikolaev, Y A; Martinac, B

    2015-09-01

    Biological membranes are essential for normal function and regulation of cells, forming a physical barrier between extracellular and intracellular space and cellular compartments. These physical barriers are subject to mechanical stresses. As a consequence, nature has developed proteins that are able to transpose mechanical stimuli into meaningful intracellular signals. These proteins, termed Mechanosensitive (MS) proteins provide a variety of roles in response to these stimuli. In prokaryotes these proteins form transmembrane spanning channels that function as osmotically activated nanovalves to prevent cell lysis by hypoosmotic shock. In eukaryotes, the function of MS proteins is more diverse and includes physiological processes such as touch, pain and hearing. The transmembrane portion of these channels is influenced by the physical properties such as charge, shape, thickness and stiffness of the lipid bilayer surrounding it, as well as the bilayer pressure profile. In this review we provide an overview of the progress to date on advances in our understanding of the intimate biophysical and chemical interactions between the lipid bilayer and mechanosensitive membrane channels, focusing on current progress in both eukaryotic and prokaryotic systems. These advances are of importance due to the increasing evidence of the role the MS channels play in disease, such as xerocytosis, muscular dystrophy and cardiac hypertrophy. Moreover, insights gained from lipid-protein interactions of MS channels are likely relevant not only to this class of membrane proteins, but other bilayer embedded proteins as well. This article is part of a Special Issue entitled: Lipid-protein interactions. PMID:25922225

  4. Efficient detection of RNA–protein interactions using tethered RNAs

    PubMed Central

    Loiselle, David; Haystead, Timothy A.; Macara, Ian G.

    2011-01-01

    The diverse localization of transcripts in cells suggests that there are many specific RNA–protein interactions that have yet to be identified. Progress has been limited, however, by the lack of a robust method to detect and isolate the RNA-binding proteins. Here we describe the use of an RNA aptamer, scaffolded to a tRNA, to create an affinity matrix that efficiently pulls down transcript-specific RNA-binding proteins from cell lysates. The addition of the tRNA scaffold to a Streptavidin aptamer (tRSA) increased binding efficiency by ∼10-fold. The tRSA system with an attached G-quartet sequence also could efficiently and specifically capture endogenous Fragile X Mental Retardation Protein (FMRP), which recognizes this RNA sequence. An alternative method, using biotinylated RNA, captured FMRP less efficiently than did our tRSA method. Finally we demonstrate the identification of novel RNA-binding proteins that interact with intron2 or 3′-UTR of the polarity protein Crumbs3 transcript. Proteins captured by these RNA sequences attached to the tRNA scaffold were identified by mass spectrometry. GFP-tagged versions of these proteins also showed specific interaction with either the Crb3 intron2 or 3′-UTR. Our tRSA technique should find wide application in mapping the RNA–protein interactome. PMID:21300640

  5. Targeting Protein-Protein Interactions with Trimeric Ligands: High Affinity Inhibitors of the MAGUK Protein Family

    PubMed Central

    Nissen, Klaus B.; Haugaard-Kedström, Linda M.; Wilbek, Theis S.; Nielsen, Line S.; Åberg, Emma; Kristensen, Anders S.; Bach, Anders; Jemth, Per; Strømgaard, Kristian

    2015-01-01

    PDZ domains in general, and those of PSD-95 in particular, are emerging as promising drug targets for diseases such as ischemic stroke. We have previously shown that dimeric ligands that simultaneously target PDZ1 and PDZ2 of PSD-95 are highly potent inhibitors of PSD-95. However, PSD-95 and the related MAGUK proteins contain three consecutive PDZ domains, hence we envisioned that targeting all three PDZ domains simultaneously would lead to more potent and potentially more specific interactions with the MAGUK proteins. Here we describe the design, synthesis and characterization of a series of trimeric ligands targeting all three PDZ domains of PSD-95 and the related MAGUK proteins, PSD-93, SAP-97 and SAP-102. Using our dimeric ligands targeting the PDZ1-2 tandem as starting point, we designed novel trimeric ligands by introducing a PDZ3-binding peptide moiety via a cysteine-derivatized NPEG linker. The trimeric ligands generally displayed increased affinities compared to the dimeric ligands in fluorescence polarization binding experiments and optimized trimeric ligands showed low nanomolar inhibition towards the four MAGUK proteins, thus being the most potent inhibitors described. Kinetic experiments using stopped-flow spectrometry showed that the increase in affinity is caused by a decrease in the dissociation rate of the trimeric ligand as compared to the dimeric ligands, likely reflecting the lower probability of simultaneous dissociation of all three PDZ ligands. Thus, we have provided novel inhibitors of the MAGUK proteins with exceptionally high affinity, which can be used to further elucidate the therapeutic potential of these proteins. PMID:25658767

  6. Fluorescence lifetime imaging microscopy (FLIM) to quantify protein-protein interactions inside cells.

    PubMed

    Duncan, R R

    2006-11-01

    Recent developments in cellular imaging spectroscopy now permit the minimally invasive study of protein dynamics inside living cells. These advances are of interest to cell biologists, as proteins rarely act in isolation, but rather in concert with others in forming cellular machinery. Until recently, all protein interactions had to be determined in vitro using biochemical approaches: this biochemical legacy has provided cell biologists with the basis to test defined protein-protein interactions not only inside cells, but now also with high spatial resolution. These techniques can detect and quantify protein behaviours down to the single-molecule level, all inside living cells. More recent developments in TCSPC (time-correlated single-photon counting) imaging are now also driving towards being able to determine protein interaction rates with similar spatial resolution, and together, these experimental advances allow investigators to perform biochemical experiments inside living cells. PMID:17052173

  7. The Development of an Interactive Videodisc Program on Protein Synthesis.

    ERIC Educational Resources Information Center

    Hazan, Charlene Corey

    An interactive videodisk (IVD) program was developed to reinforce learning of the biological concept of protein synthesis for high school students. The laser videodisc "The Living Textbook Life Science" was the source of frames, and the authoring system of G. Smith was used to create the disc. The interactive program was designed to make the…

  8. Influence of hydrodynamic interactions on mechanical unfolding of proteins

    NASA Astrophysics Data System (ADS)

    Szymczak, P.; Cieplak, Marek

    2007-07-01

    We incorporate hydrodynamic interactions in a structure-based model of ubiquitin and demonstrate that the hydrodynamic coupling may reduce the peak force when stretching the protein at constant speed, especially at larger speeds. Hydrodynamic interactions are also shown to facilitate unfolding at constant force and inhibit stretching by fluid flows.

  9. Protein-protein interaction studies based on molecular aptamers by affinity capillary electrophoresis.

    PubMed

    Huang, Chih-Ching; Cao, Zehui; Chang, Huan-Tsung; Tan, Weihong

    2004-12-01

    Protein-DNA/protein-protein interactions play critical roles in many biological processes. We report here the investigation of protein-protein interactions using molecular aptamers with affinity capillary electrophoresis (ACE). A human alpha-thrombin binding aptamer was labeled with 6-carboxyfluorescein and exploited as a selective fluorescent probe for studying thrombin-protein interactions using capillary electrophoresis with laser-induced fluorescence. A 15-mer binding DNA aptamer can be separated into two peaks in CE that correspond to the linear aptamer (L-Apt) and the thrombin-binding G-quadruplex structure in the presence of K(+) or Ba(2+). In a bare capillary, the peak area of G-quadruplex aptamer (G-Apt) was found to decrease with the addition of thrombin while that of L-Apt remained unchanged. Even though the peak of the G-Apt/thrombin binding complex is broad due to a weaker binding affinity between aptamer and thrombin, we were still able to quantify the thrombin and anti-thrombin proteins (human anti-thrombin III, AT III) based on the peak areas of free G-Apt. The detection limits of thrombin and AT III were 9.8 and 2.1 nM, respectively. The aptamer-based competitive ACE assay has also been applied to quantify thrombin-anti-thrombin III interaction and to monitor this reaction in real time. The addition of poly(ethylene glycol) to the sample matrix stabilized the complex of the G-Aptthrombin. This assay can be used to study the interactions between thrombin and proteins that do not disrupt G-Apt binding property at Exosit I site of the thrombin. Our aptamer-based ACE assay can be an effective approach for studying protein-protein interactions and for analyzing binding site and binding constant information in protein-protein and protein-DNA interaction studies. PMID:15571349

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

    PubMed

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

    2016-08-01

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

  11. Sequence-based prediction of protein-protein interaction sites with L1-logreg classifier.

    PubMed

    Dhole, Kaustubh; Singh, Gurdeep; Pai, Priyadarshini P; Mondal, Sukanta

    2014-05-01

    Protein-protein interactions are of central importance for virtually every process in a living cell. Information about the interaction sites in proteins improves our understanding of disease mechanisms and can provide the basis for new therapeutic approaches. Since a multitude of unique residue-residue contacts facilitate the interactions, protein-protein interaction sites prediction has become one of the most important and challenging problems of computational biology. Although much progress in this field has been reported, this problem is yet to be satisfactorily solved. Here, a novel method (LORIS: L1-regularized LOgistic Regression based protein-protein Interaction Sites predictor) is proposed, that identifies interaction residues, using sequence features and is implemented via the L1-logreg classifier. Results show that LORIS is not only quite effective, but also, performs better than existing state-of-the art methods. LORIS, available as standalone package, can be useful for facilitating drug-design and targeted mutation related studies, which require a deeper knowledge of protein interactions sites. PMID:24486250

  12. Protein-protein interaction between ezrin and p65 in human breast cancer cells.

    PubMed

    Tang, R; Li, F X; Shao, W F; Wen, Q S; Yu, X R; Xiong, J B

    2016-01-01

    Our study aimed to investigate the co-localization and protein-protein interactions between ezrin and p65 in human breast cancer cells. Liquid chromatography-mass spectrometry (LCMS) was used to uncover novel protein interactions with ezrin in MDA-MB-231 cells. Endogenous co-immunoprecipitation was used to validate protein-protein interactions between ezrin and p65 in MDA-MB-231. Exogenous interactions between ezrin and p65 were validated in MDA-MB-231 cells via Flag-ezrin and HA-p65 co-transfection and followed by co-immunoprecipitation. Immunofluorescence staining was used to visualize ezrin and p65 co-localization in MDA-MB-231. LCMS results showed that there were 1000 proteins interacting with ezrin in MDA-MB-231 cells. Ezrin and p65 interactions were confirmed with both endogenous and exogenous methods. We were also able to visualize ezrin and p65 co-localization in MDA-MB-231. In summary, we found protein-protein interactions between Ezrin and p65 in human breast cancer cells. PMID:27420986

  13. Homology Inference of Protein-Protein Interactions via Conserved Binding Sites

    PubMed Central

    Tyagi, Manoj; Thangudu, Ratna R.; Zhang, Dachuan; Bryant, Stephen H.; Madej, Thomas; Panchenko, Anna R.

    2012-01-01

    The coverage and reliability of protein-protein interactions determined by high-throughput experiments still needs to be improved, especially for higher organisms, therefore the question persists, how interactions can be verified and predicted by computational approaches using available data on protein structural complexes. Recently we developed an approach called IBIS (Inferred Biomolecular Interaction Server) to predict and annotate protein-protein binding sites and interaction partners, which is based on the assumption that the structural location and sequence patterns of protein-protein binding sites are conserved between close homologs. In this study first we confirmed high accuracy of our method and found that its accuracy depends critically on the usage of all available data on structures of homologous complexes, compared to the approaches where only a non-redundant set of complexes is employed. Second we showed that there exists a trade-off between specificity and sensitivity if we employ in the prediction only evolutionarily conserved binding site clusters or clusters supported by only one observation (singletons). Finally we addressed the question of identifying the biologically relevant interactions using the homology inference approach and demonstrated that a large majority of crystal packing interactions can be correctly identified and filtered by our algorithm. At the same time, about half of biological interfaces that are not present in the protein crystallographic asymmetric unit can be reconstructed by IBIS from homologous complexes without the prior knowledge of crystal parameters of the query protein. PMID:22303436

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

    PubMed Central

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

    2011-01-01

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

  15. Mechanisms of Peroxynitrite Interactions with Heme Proteins

    PubMed Central

    Su, Jia; Groves, John T.

    2010-01-01

    Oxygenated hemoproteins are known to react rapidly with nitric oxide (NO) to produce peroxynitrite (PN) at the heme site. This process could lead either to attenuation of the effects of NO or to nitrosative protein damage. Peroxynitrite is a powerful nitrating and oxidizing agent that has been implicated in a variety of cell injuries. Accordingly, it is important to delineate the nature and variety of reaction mechanisms of PN reactions with heme proteins. In this Forum we survey the range of reactions of PN with heme proteins, with particular attention to myoglobin and cytochrome c. While these two proteins are textbook paradigms for oxygen binding and electron transfer, respectively, both have recently been shown to have other important functions that involve nitric oxide and peroxynitrite. We have recently described direct evidence that ferrylMb and NO2 are both produced during the reaction of PN and metmyolgobin (metMb). Kinetic evidence indicates that these products evolve from initial formation of a caged radical intermediate [FeIV=O .NO2]. This caged pair reacts mainly via internal return with a rate constant kr to form metMb and nitrate in an oxygen rebound scenario. Detectable amounts of ferrylMb are observed by stopped-flow spectrophotometry, appearing at a rate consistent with the rate, kobs of heme-mediated PN decomposition. Freely-diffusing NO2, which is liberated concomitantly from the radical pair (ke), preferentially nitrates myoglobin Tyr103 and added fluorescein. For cytochrome c, Raman spectroscopy has revealed that a substantial fraction of cytochrome c converts to a β-sheet structure, at the expense of turns and helices at low pH. It is proposed that a short β-sheet segment, comprising residues 37-39 and 58-61, extends itself into the large 37-61 loop when the latter is destabilized by protonation of H26, which forms an anchoring H-bond to loop residue P44. This conformation change ruptures the Met80-Fe bond, as revealed by changes in

  16. Protein-protein interaction inference based on semantic similarity of Gene Ontology terms.

    PubMed

    Zhang, Shu-Bo; Tang, Qiang-Rong

    2016-07-21

    Identifying protein-protein interactions is important in molecular biology. Experimental methods to this issue have their limitations, and computational approaches have attracted more and more attentions from the biological community. The semantic similarity derived from the Gene Ontology (GO) annotation has been regarded as one of the most powerful indicators for protein interaction. However, conventional methods based on GO similarity fail to take advantage of the specificity of GO terms in the ontology graph. We proposed a GO-based method to predict protein-protein interaction by integrating different kinds of similarity measures derived from the intrinsic structure of GO graph. We extended five existing methods to derive the semantic similarity measures from the descending part of two GO terms in the GO graph, then adopted a feature integration strategy to combines both the ascending and the descending similarity scores derived from the three sub-ontologies to construct various kinds of features to characterize each protein pair. Support vector machines (SVM) were employed as discriminate classifiers, and five-fold cross validation experiments were conducted on both human and yeast protein-protein interaction datasets to evaluate the performance of different kinds of integrated features, the experimental results suggest the best performance of the feature that combines information from both the ascending and the descending parts of the three ontologies. Our method is appealing for effective prediction of protein-protein interaction. PMID:27117309

  17. Interaction of the tobacco lectin with histone proteins.

    PubMed

    Schouppe, Dieter; Ghesquière, Bart; Menschaert, Gerben; De Vos, Winnok H; Bourque, Stéphane; Trooskens, Geert; Proost, Paul; Gevaert, Kris; Van Damme, Els J M

    2011-03-01

    The tobacco (Nicotiana tabacum) agglutinin or Nictaba is a member of a novel class of plant lectins residing in the nucleus and the cytoplasm of tobacco cells. Since tobacco lectin expression is only observed after the plant has been subjected to stress situations such as jasmonate treatment or insect attack, Nictaba is believed to act as a signaling protein involved in the stress physiology of the plant. In this paper, a nuclear proteomics approach was followed to identify the binding partners for Nictaba in the nucleus and the cytoplasm of tobacco cv Xanthi cells. Using lectin affinity chromatography and pull-down assays, it was shown that Nictaba interacts primarily with histone proteins. Binding of Nictaba with histone H2B was confirmed in vitro using affinity chromatography of purified calf thymus histone proteins on a Nictaba column. Elution of Nictaba-interacting histone proteins was achieved with 1 m N-acetylglucosamine (GlcNAc). Moreover, mass spectrometry analyses indicated that the Nictaba-interacting histone proteins are modified by O-GlcNAc. Since the lectin-histone interaction was shown to be carbohydrate dependent, it is proposed that Nictaba might fulfill a signaling role in response to stress by interacting with O-GlcNAcylated proteins in the plant cell nucleus. PMID:21224338

  18. Interaction of ion channels and receptors with PDZ domain proteins.

    PubMed

    Kornau, H C; Seeburg, P H; Kennedy, M B

    1997-06-01

    The complex anatomy of neurons demands a high degree of functional organization. Therefore, membrane receptors and ion channels are often localized to selected subcellular sites and coupled to specific signal transduction machineries. PDZ domains have come into focus as protein interaction modules that mediate the binding of a class of submembraneous proteins to membrane receptors and ion channels and thus subserve these organizational aspects. The structures of two PDZ domains have been resolved, which has led to a structural understanding of the specificity of interactions of various PDZ domains with their respective partners. The functional implications of PDZ domain interactions are now being addressed in vitro and in vivo. PMID:9232802

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

    PubMed Central

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

    2014-01-01

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

  20. Protein-lipid interactions: paparazzi hunting for snap-shots.

    PubMed

    Haberkant, Per; van Meer, Gerrit

    2009-08-01

    Photoactivatable groups meeting the criterion of minimal perturbance allow the investigation of interactions in biological samples. Here, we review the application of photoactivatable groups in lipids enabling the study of protein-lipid interactions in (biological) membranes. The chemistry of various photoactivatable groups is summarized and the specificity of the interactions detected is discussed. The recent introduction of 'click chemistry' in photocrosslinking of membrane proteins by photo-activatable lipids opens new possibilities for the analysis of crosslinked products and will help to close the gap between proteomics and lipidomics. PMID:19426134

  1. High Throughput Screening Method to Explore Protein Interactions with Nanoparticles.

    PubMed

    Nasir, Irem; Fatih, Warda; Svensson, Anja; Radu, Dennis; Linse, Sara; Cabaleiro Lago, Celia; Lundqvist, Martin

    2015-01-01

    The interactions of biological macromolecules with nanoparticles underlie a wide variety of current and future applications in the fields of biotechnology, medicine and bioremediation. The same interactions are also responsible for mediating potential biohazards of nanomaterials. Some applications require that proteins adsorb to the nanomaterial and that the protein resists or undergoes structural rearrangements. This article presents a screening method for detecting nanoparticle-protein partners and conformational changes on time scales ranging from milliseconds to days. Mobile fluorophores are used as reporters to study the interaction between proteins and nanoparticles in a high-throughput manner in multi-well format. Furthermore, the screening method may reveal changes in colloidal stability of nanomaterials depending on the physicochemical conditions. PMID:26313757

  2. High Throughput Screening Method to Explore Protein Interactions with Nanoparticles

    PubMed Central

    Nasir, Irem; Fatih, Warda; Svensson, Anja; Radu, Dennis; Linse, Sara; Cabaleiro Lago, Celia; Lundqvist, Martin

    2015-01-01

    The interactions of biological macromolecules with nanoparticles underlie a wide variety of current and future applications in the fields of biotechnology, medicine and bioremediation. The same interactions are also responsible for mediating potential biohazards of nanomaterials. Some applications require that proteins adsorb to the nanomaterial and that the protein resists or undergoes structural rearrangements. This article presents a screening method for detecting nanoparticle-protein partners and conformational changes on time scales ranging from milliseconds to days. Mobile fluorophores are used as reporters to study the interaction between proteins and nanoparticles in a high-throughput manner in multi-well format. Furthermore, the screening method may reveal changes in colloidal stability of nanomaterials depending on the physicochemical conditions. PMID:26313757

  3. Protein-surface interactions on stimuli-responsive polymeric biomaterials.

    PubMed

    Cross, Michael C; Toomey, Ryan G; Gallant, Nathan D

    2016-04-01

    Responsive surfaces: a review of the dependence of protein adsorption on the reversible volume phase transition in stimuli-responsive polymers. Specifically addressed are a widely studied subset: thermoresponsive polymers. Findings are also generalizable to other materials which undergo a similarly reversible volume phase transition. As of 2015, over 100,000 articles have been published on stimuli-responsive polymers and many more on protein-biomaterial interactions. Significantly, fewer than 100 of these have focused specifically on protein interactions with stimuli-responsive polymers. These report a clear trend of increased protein adsorption in the collapsed state compared to the swollen state. This control over protein interactions makes stimuli-responsive polymers highly useful in biomedical applications such as wound repair scaffolds, on-demand drug delivery, and antifouling surfaces. Outstanding questions are whether the protein adsorption is reversible with the volume phase transition and whether there is a time-dependence. A clear understanding of protein interactions with stimuli-responsive polymers will advance theoretical models, experimental results, and biomedical applications. PMID:26942693

  4. Structural studies of lipid-protein interactions on cushioned bilayers

    NASA Astrophysics Data System (ADS)

    Ghosh, S. K.; Mukhopadhyay, M. K.; Ma, Y.; Lopez, I.; Bera, S.; Lurio, L. B.; Chakrabarti, A.; Kim, J. E.; Sanyal, M. K.; Sinha, S. K.

    2013-03-01

    Biological membranes are heterogeneous and dynamical organizations of lipids and proteins, which perform functions fundamental to cell survival. Lipid-protein interactions control these functions by influencing folding and stability of integral or peripheral membrane proteins. Further, the incorporation or adsorption of these proteins into the membrane can in turn influence the lipid bilayer properties. In spite of some progress in understanding this process, a detailed structural analysis is lacking. Towards a better understanding of this interaction, we have performed an advanced interface sensitive scattering experiment using synchrotron x-rays. To accurately mimic the biological membranes with their natural thermal fluctuations and in-plane mobility of lipid molecules, polymer cushioned lipid bilayers have been used. This study shows that the adsorption of peripheral membrane proteinspectrindepends on the lipid headgroups, exhibiting different types of binding to phosphatidylcholine (PC) and phosphatidylethanolamie (PE). Further, the interaction of outer membrane protein A (OMP-A), an integral membrane protein is sensitive to the thermodynamic phase of the lipids. A detailed physical modeling of the lipid-protein interactions is under way.

  5. On the surface interactions of proteins with lignin.

    PubMed

    Salas, Carlos; Rojas, Orlando J; Lucia, Lucian A; Hubbe, Martin A; Genzer, Jan

    2013-01-01

    Lignins are used often in formulations involving proteins but little is known about the surface interactions between these important biomacromolecules. In this work, we investigate the interactions at the solid-liquid interface of lignin with the two main proteins in soy, glycinin (11S) and β-conglycinin (7S). The extent of adsorption of 11S and 7S onto lignin films and the degree of hydration of the interfacial layers is quantified via Quartz crystal microgravimetry (QCM) and surface plasmon resonance (SPR). Solution ionic strength and protein denaturation (2-mercaptoethanol and urea) critically affect the adsorption process as protein molecules undergo conformational changes and their hydrophobic or hydrophilic amino acid residues interact with the surrounding medium. In general, the adsorption of the undenatured proteins onto lignin is more extensive compared to that of the denatured biomolecules and a large amount of water is coupled to the adsorbed molecules. The reduction in water contact angle after protein adsorption (by ~40° and 35° for undenatured 11S and 7S, respectively) is explained by strong nonspecific interactions between soy proteins and lignin. PMID:23234476

  6. A selection that reports on protein-protein interactions within a thermophilic bacterium.

    PubMed

    Nguyen, Peter Q; Silberg, Jonathan J

    2010-07-01

    Many proteins can be split into fragments that exhibit enhanced function upon fusion to interacting proteins. While this strategy has been widely used to create protein-fragment complementation assays (PCAs) for discovering protein-protein interactions within mesophilic organisms, similar assays have not yet been developed for studying natural and engineered protein complexes at the temperatures where thermophilic microbes grow. We describe the development of a selection for protein-protein interactions within Thermus thermophilus that is based upon growth complementation by fragments of Thermotoga neapolitana adenylate kinase (AK(Tn)). Complementation studies with an engineered thermophile (PQN1) that is not viable above 75 degrees C because its adk gene has been replaced by a Geobacillus stearothermophilus ortholog revealed that growth could be restored at 78 degrees C by a vector that coexpresses polypeptides corresponding to residues 1-79 and 80-220 of AK(Tn). In contrast, PQN1 growth was not complemented by AK(Tn) fragments harboring a C156A mutation within the zinc-binding tetracysteine motif unless these fragments were fused to Thermotoga maritima chemotaxis proteins that heterodimerize (CheA and CheY) or homodimerize (CheX). This enhanced complementation is interpreted as arising from chemotaxis protein-protein interactions, since AK(Tn)-C156A fragments having only one polypeptide fused to a chemotaxis protein did not complement PQN1 to the same extent. This selection increases the maximum temperature where a PCA can be used to engineer thermostable protein complexes and to map protein-protein interactions. PMID:20418388

  7. Optoelectronic Tweezers Integrated with Lens-free Holographic Microscopy for Wide-field Interactive Cell and Particle Manipulation on a Chip

    PubMed Central

    Huang, Kuo-Wei; Su, Ting-Wei; Ozcan, Aydogan; Chiou, Pei-Yu

    2013-01-01

    We demonstrate an optoelectronic tweezer (OET) coupled to a lensfree holographic microscope for real-time interactive manipulation of cells and micro-particles over a large field-of-view (FOV). This integrated platform can record the holographic images of cells and particles over the entire active area of a CCD sensor array, perform digital image reconstruction to identify target cells, dynamically track the positions of cells and particles, and project light beams to trigger light-induced dielectrophoretic forces to pattern and sort cells on a chip. OET technology has been previously shown capable of performing parallel single cell manipulation over a large area. However, its throughput has been bottlenecked by the number of cells that can be imaged within the limited FOV of a conventional microscope objective lens. Integrating lensfree holographic imaging with OET solves this fundamental FOV barrier, while also creating a compact on-chip cell/particle manipulation platform. Using this unique platform, we have successfully demonstrated real-time interactive manipulation of thousands of single cells and micro-particles over an ultra-large area of e.g., 240 mm2 (i.e. 17.96 mm × 13.52 mm). PMID:23661233

  8. Evidence for the interaction of the regulatory protein Ki-1/57 with p53 and its interacting proteins

    SciTech Connect

    Nery, Flavia C.; Rui, Edmilson; Kuniyoshi, Tais M.; Kobarg, Joerg . E-mail: jkobarg@lnls.br

    2006-03-17

    Ki-1/57 is a cytoplasmic and nuclear phospho-protein of 57 kDa and interacts with the adaptor protein RACK1, the transcription factor MEF2C, and the chromatin remodeling factor CHD3, suggesting that it might be involved in the regulation of transcription. Here, we describe yeast two-hybrid studies that identified a total of 11 proteins interacting with Ki-1/57, all of which interact or are functionally associated with p53 or other members of the p53 family of proteins. We further found that Ki-1/57 is able to interact with p53 itself in the yeast two-hybrid system when the interaction was tested directly. This interaction could be confirmed by pull down assays with purified proteins in vitro and by reciprocal co-immunoprecipitation assays from the human Hodgkin analogous lymphoma cell line L540. Furthermore, we found that the phosphorylation of p53 by PKC abolishes its interaction with Ki-1/57 in vitro.

  9. Identification of a novel protein-protein interaction motif mediating interaction of GPCR-associated sorting proteins with G protein-coupled receptors.

    PubMed

    Bornert, Olivier; Møller, Thor C; Boeuf, Julien; Candusso, Marie-Pierre; Wagner, Renaud; Martinez, Karen L; Simonin, Frederic

    2013-01-01

    GPCR desensitization and down-regulation are considered key molecular events underlying the development of tolerance in vivo. Among the many regulatory proteins that are involved in these complex processes, GASP-1 have been shown to participate to the sorting of several receptors toward the degradation pathway. This protein belongs to the recently identified GPCR-associated sorting proteins (GASPs) family that comprises ten members for which structural and functional details are poorly documented. We present here a detailed structure-function relationship analysis of the molecular interaction between GASPs and a panel of GPCRs. In a first step, GST-pull down experiments revealed that all the tested GASPs display significant interactions with a wide range of GPCRs. Importantly, the different GASP members exhibiting the strongest interaction properties were also characterized by the presence of a small, highly conserved and repeated "GASP motif" of 15 amino acids. We further showed using GST-pull down, surface plasmon resonance and co-immunoprecipitation experiments that the central domain of GASP-1, which contains 22 GASP motifs, is essential for the interaction with GPCRs. We then used site directed mutagenesis and competition experiments with synthetic peptides to demonstrate that the GASP motif, and particularly its highly conserved core sequence SWFW, is critically involved in the interaction with GPCRs. Overall, our data show that several members of the GASP family interact with GPCRs and highlight the presence within GASPs of a novel protein-protein interaction motif that might represent a new target to investigate the involvement of GASPs in the modulation of the activity of GPCRs. PMID:23441177

  10. Interactions between RNase P protein subunits in Archaea

    PubMed Central

    Hall, Thomas A.; Brown, James W.

    2004-01-01

    A yeast two-hybrid system was used to identify protein–protein interactions between the ribonuclease P (RNase P) protein subunits Mth11p, Mth687p, Mth688p and Mth1618p from the archaeon Methanothermobacter thermoautotrophicus. Clear interactions between Mth688p and Mth687p, and between Mth1618p and Mth11p, were confirmed by HIS3 and LacZ reporter expression. Weaker interactions of Mth687p and Mth688p with Mth11p, and Mth11p with itself, are also suggested. These interactions resemble, and confirm, those previously seen among the homologs of these proteins in the more complex yeast RNase P holoenzyme. PMID:15810434

  11. MEGADOCK: an all-to-all protein-protein interaction prediction system using tertiary structure data.

    PubMed

    Ohue, Masahito; Matsuzaki, Yuri; Uchikoga, Nobuyuki; Ishida, Takashi; Akiyama, Yutaka

    2014-01-01

    The elucidation of protein-protein interaction (PPI) networks is important for understanding cellular structure and function and structure-based drug design. However, the development of an effective method to conduct exhaustive PPI screening represents a computational challenge. We have been investigating a protein docking approach based on shape complementarity and physicochemical properties. We describe here the development of the protein-protein docking software package "MEGADOCK" that samples an extremely large number of protein dockings at high speed. MEGADOCK reduces the calculation time required for docking by using several techniques such as a novel scoring function called the real Pairwise Shape Complementarity (rPSC) score. We showed that MEGADOCK is capable of exhaustive PPI screening by completing docking calculations 7.5 times faster than the conventional docking software, ZDOCK, while maintaining an acceptable level of accuracy. When MEGADOCK was applied to a subset of a general benchmark dataset to predict 120 relevant interacting pairs from 120 x 120 = 14,400 combinations of proteins, an F-measure value of 0.231 was obtained. Further, we showed that MEGADOCK can be applied to a large-scale protein-protein interaction-screening problem with accuracy better than random. When our approach is combined with parallel high-performance computing systems, it is now feasible to search and analyze protein-protein interactions while taking into account three-dimensional structures at the interactome scale. MEGADOCK is freely available at http://www.bi.cs.titech.ac.jp/megadock. PMID:23855673

  12. Molecular tweezers modulate 14-3-3 protein-protein interactions

    NASA Astrophysics Data System (ADS)

    Bier, David; Rose, Rolf; Bravo-Rodriguez, Kenny; Bartel, Maria; Ramirez-Anguita, Juan Manuel; Dutt, Som; Wilch, Constanze; Klärner, Frank-Gerrit; Sanchez-Garcia, Elsa; Schrader, Thomas; Ottmann, Christian

    2013-03-01

    Supramolecular chemistry has recently emerged as a promising way to modulate protein functions, but devising molecules that will interact with a protein in the desired manner is difficult as many competing interactions exist in a biological environment (with solvents, salts or different sites for the target biomolecule). We now show that lysine-specific molecular tweezers bind to a 14-3-3 adapter protein and modulate its interaction with partner proteins. The tweezers inhibit binding between the 14-3-3 protein and two partner proteins—a phosphorylated (C-Raf) protein and an unphosphorylated one (ExoS)—in a concentration-dependent manner. Protein crystallography shows that this effect arises from the binding of the tweezers to a single surface-exposed lysine (Lys214) of the 14-3-3 protein in the proximity of its central channel, which normally binds the partner proteins. A combination of structural analysis and computer simulations provides rules for the tweezers' binding preferences, thus allowing us to predict their influence on this type of protein-protein interactions.

  13. Multi-level machine learning prediction of protein-protein interactions in Saccharomyces cerevisiae.

    PubMed

    Zubek, Julian; Tatjewski, Marcin; Boniecki, Adam; Mnich, Maciej; Basu, Subhadip; Plewczynski, Dariusz

    2015-01-01

    Accurate identification of protein-protein interactions (PPI) is the key step in understanding proteins' biological functions, which are typically context-dependent. Many existing PPI predictors rely on aggregated features from protein sequences, however only a few methods exploit local information about specific residue contacts. In this work we present a two-stage machine learning approach for prediction of protein-protein interactions. We start with the carefully filtered data on protein complexes available for Saccharomyces cerevisiae in the Protein Data Bank (PDB) database. First, we build linear descriptions of interacting and non-interacting sequence segment pairs based on their inter-residue distances. Secondly, we train machine learning classifiers to predict binary segment interactions for any two short sequence fragments. The final prediction of the protein-protein interaction is done using the 2D matrix representation of all-against-all possible interacting sequence segments of both analysed proteins. The level-I predictor achieves 0.88 AUC for micro-scale, i.e., residue-level prediction. The level-II predictor improves the results further by a more complex learning paradigm. We perform 30-fold macro-scale, i.e., protein-level cross-validation experiment. The level-II predictor using PSIPRED-predicted secondary structure reaches 0.70 precision, 0.68 recall, and 0.70 AUC, whereas other popular methods provide results below 0.6 threshold (recall, precision, AUC). Our results demonstrate that multi-scale sequence features aggregation procedure is able to improve the machine learning results by more than 10% as compared to other sequence representations. Prepared datasets and source code for our experimental pipeline are freely available for download from: http://zubekj.github.io/mlppi/ (open source Python implementation, OS independent). PMID:26157620

  14. Uncovering New Pathogen–Host Protein–Protein Interactions by Pairwise Structure Similarity

    PubMed Central

    Cui, Tao; Li, Weihui; Liu, Lei; Huang, Qiaoyun; He, Zheng-Guo

    2016-01-01

    Pathogens usually evade and manipulate host-immune pathways through pathogen–host protein–protein interactions (PPIs) to avoid being killed by the host immune system. Therefore, uncovering pathogen–host PPIs is critical for determining the mechanisms underlying pathogen infection and survival. In this study, we developed a computational method, which we named pairwise structure similarity (PSS)-PPI, to predict pathogen–host PPIs. First, a high-quality and non-redundant structure–structure interaction (SSI) template library was constructed by exhaustively exploring heteromeric protein complex structures in the PDB database. New interactions were then predicted by searching for PSS with complex structures in the SSI template library. A quantitative score named the PSS score, which integrated structure similarity and residue–residue contact-coverage information, was used to describe the overall similarity of each predicted interaction with the corresponding SSI template. Notably, PSS-PPI yielded experimentally confirmed pathogen–host PPIs of human immunodeficiency virus type 1 (HIV-1) with performance close to that of in vitro high-throughput screening approaches. Finally, a pathogen–host PPI network of human pathogen Mycobacterium tuberculosis, the causative agent of tuberculosis, was constructed using PSS-PPI and refined using filtration steps based on cellular localization information. Analysis of the resulting network indicated that secreted proteins of the STPK, ESX-1, and PE/PPE family in M. tuberculosis targeted human proteins involved in immune response and phagocytosis. M. tuberculosis also targeted host factors known to regulate HIV replication. Taken together, our findings provide insights into the survival mechanisms of M. tuberculosis in human hosts, as well as co-infection of tuberculosis and HIV. With the rapid pace of three-dimensional protein structure discovery, the SSI template library we constructed and the PSS-PPI method we devised

  15. Predicting direct protein interactions from affinity purification mass spectrometry data

    PubMed Central

    2010-01-01

    Background Affinity purification followed by mass spectrometry identification (AP-MS) is an increasingly popular approach to observe protein-protein interactions (PPI) in vivo. One drawback of AP-MS, however, is that it is prone to detecting indirect interactions mixed with direct physical interactions. Therefore, the ability to distinguish direct interactions from indirect ones is of much interest. Results We first propose a simple probabilistic model for the interactions captured by AP-MS experiments, under which the problem of separating direct interactions from indirect ones is formulated. Then, given idealized quantitative AP-MS data, we study the problem of identifying the most likely set of direct interactions that produced the observed data. We address this challenging graph theoretical problem by first characterizing signatures that can identify weakly connected nodes as well as dense regions of the network. The rest of the direct PPI network is then inferred using a genetic algorithm. Our algorithm shows good performance on both simulated and biological networks with very high sensitivity and specificity. Then the algorithm is used to predict direct interactions from a set of AP-MS PPI data from yeast, and its performance is measured against a high-quality interaction dataset. Conclusions As the sensitivity of AP-MS pipeline improves, the fraction of indirect interactions detected will also increase, thereby making the ability to distinguish them even more desirable. Despite the simplicity of our model for indirect interactions, our method provides a good performance on the test networks. PMID:21034440

  16. Analysis of interactions between ribosomal proteins and RNA structural motifs

    PubMed Central

    2010-01-01

    Background One important goal of structural bioinformatics is to recognize and predict the interactions between protein binding sites and RNA. Recently, a comprehensive analysis of ribosomal proteins and their interactions with rRNA has been done. Interesting results emerged from the comparison of r-proteins within the small subunit in T. thermophilus and E. coli, supporting the idea of a core made by both RNA and proteins, conserved by evolution. Recent work showed also that ribosomal RNA is modularly composed. Motifs are generally single-stranded sequences of consecutive nucleotides (ssRNA) with characteristic folding. The role of these motifs in protein-RNA interactions has been so far only sparsely investigated. Results This work explores the role of RNA structural motifs in the interaction of proteins with ribosomal RNA (rRNA). We analyze composition, local geometries and conformation of interface regions involving motifs such as tetraloops, kink turns and single extruded nucleotides. We construct an interaction map of protein binding sites that allows us to identify the common types of shared 3-D physicochemical binding patterns for tetraloops. Furthermore, we investigate the protein binding pockets that accommodate single extruded nucleotides either involved in kink-turns or in arbitrary RNA strands. This analysis reveals a new structural motif, called tripod. It corresponds to small pockets consisting of three aminoacids arranged at the vertices of an almost equilateral triangle. We developed a search procedure for the recognition of tripods, based on an empirical tripod fingerprint. Conclusion A comparative analysis with the overall RNA surface and interfaces shows that contact surfaces involving RNA motifs have distinctive features that may be useful for the recognition and prediction of interactions. PMID:20122215

  17. A protein interaction map for cell polarity development

    PubMed Central

    Drees, Becky L.; Sundin, Bryan; Brazeau, Elizabeth; Caviston, Juliane P.; Chen, Guang-Chao; Guo, Wei; Kozminski, Keith G.; Lau, Michelle W.; Moskow, John J.; Tong, Amy; Schenkman, Laura R.; McKenzie, Amos; Brennwald, Patrick; Longtine, Mark; Bi, Erfei; Chan, Clarence; Novick, Peter; Boone, Charles; Pringle, John R.; Davis, Trisha N.; Fields, Stanley; Drubin, David G.

    2001-01-01

    Many genes required for cell polarity development in budding yeast have been identified and arranged into a functional hierarchy. Core elements of the hierarchy are widely conserved, underlying cell polarity development in diverse eukaryotes. To enumerate more fully the protein–protein interactions that mediate cell polarity development, and to uncover novel mechanisms that coordinate the numerous events involved, we carried out a large-scale two-hybrid experiment. 68 Gal4 DNA binding domain fusions of yeast proteins associated with the actin cytoskeleton, septins, the secretory apparatus, and Rho-type GTPases were used to screen an array of yeast transformants that express ∼90% of the predicted Saccharomyces cerevisiae open reading frames as Gal4 activation domain fusions. 191 protein–protein interactions were detected, of which 128 had not been described previously. 44 interactions implicated 20 previously uncharacterized proteins in cell polarity development. Further insights into possible roles of 13 of these proteins were revealed by their multiple two-hybrid interactions and by subcellular localization. Included in the interaction network were associations of Cdc42 and Rho1 pathways with proteins involved in exocytosis, septin organization, actin assembly, microtubule organization, autophagy, cytokinesis, and cell wall synthesis. Other interactions suggested direct connections between Rho1- and Cdc42-regulated pathways; the secretory apparatus and regulators of polarity establishment; actin assembly and the morphogenesis checkpoint; and the exocytic and endocytic machinery. In total, a network of interactions that provide an integrated response of signaling proteins, the cytoskeleton, and organelles to the spatial cues that direct polarity development was revealed. PMID:11489916

  18. Identification of novel CBP interacting proteins in embryonic orofacial tissue

    SciTech Connect

    Yin Xiaolong; Warner, Dennis R.; Roberts, Emily A.; Pisano, M. Michele; Greene, Robert M. . E-mail: greene@louisville.edu

    2005-04-15

    cAMP response element-binding protein (CREB)-binding protein (CBP) plays an important role as a general co-integrator of multiple signaling pathways and interacts with a large number of transcription factors and co-factors, through its numerous protein-binding domains. To identify nuclear factors associated with CBP in developing orofacial tissue, a yeast two-hybrid screen of a cDNA library derived from orofacial tissue from gestational day 11 to 13 mouse embryos was conducted. Using the carboxy terminus (amino acid residues 1676-2441) of CBP as bait, several novel proteins that bind CBP were identified, including an Msx-interacting-zinc finger protein, CDC42 interaction protein 4/thyroid hormone receptor interactor 10, SH3-domain GRB2-like 1, CCR4-NOT transcription complex subunit 3, adaptor protein complex AP-1 {beta}1 subunit, eukaryotic translation initiation factor 2B subunit 1 ({alpha}), and cyclin G-associated kinase. Results of the yeast two-hybrid screen were confirmed by glutathione S-transferase pull-down assays. The identification of these proteins as novel CBP-binding partners allows exploration of new mechanisms by which CBP regulates and integrates diverse cell signaling pathways.

  19. Screening for Host Factors Directly Interacting with RSV Protein: Microfluidics.

    PubMed

    Kipper, Sarit; Avrahami, Dorit; Bajorek, Monika; Gerber, Doron

    2016-01-01

    We present a high-throughput microfluidics platform to identify novel host cell binding partners of respiratory syncytial virus (RSV) matrix (M) protein. The device consists of thousands of reaction chambers controlled by micro-mechanical valves. The microfluidic device is mated to a microarray-printed custom-made gene library. These genes are then transcribed and translated on-chip, resulting in a protein array ready for binding to RSV M protein.Even small viral proteome, such as that of RSV, presents a challenge due to the fact that viral proteins are usually multifunctional and thus their interaction with the host is complex. Protein microarrays technology allows the interrogation of protein-protein interactions, which could possibly overcome obstacles by using conventional high throughput methods. Using microfluidics platform we have identified new host interactors of M involved in various cellular pathways. A number of microfluidics based assays have already provided novel insights into the virus-host interactome, and the results have important implications for future antiviral strategies aimed at targets of viral protein interactions with the host. PMID:27464694

  20. Analytical Strategies for Detecting Nanoparticle-Protein Interactions

    PubMed Central

    Li, Liwen; Mu, Qingxin; Zhang, Bin; Yan, Bing

    2016-01-01

    A significant increase in biomedical applications of nanomaterials and their potential toxicity demand versatile analytical techniques to determine protein – nanoparticle (NP) interactions. These diverse analytical techniques are reviewed. Spectroscopic methods play a significant role in studying binding affinity, binding ratio, and binding mechanisms. To elucidate NP-proteome interactions, chromatography and electrophoresis techniques are applied to separate NP-bound proteins and Matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) to identify these proteins. Since NP-protein binding is a dynamic event, Surface Plasmon Resonance (SPR) and Quartz Crystal Microbalance (QCM) are methods of choice to study kinetics of NP-protein binding. PMID:20502814

  1. Structural approaches to probing metal interaction with proteins.

    PubMed

    Parker, Lorien J; Ascher, David B; Gao, Chen; Miles, Luke A; Harris, Hugh H; Parker, Michael W

    2012-10-01

    In this mini-review we focus on metal interactions with proteins with a particular emphasis on the evident synergism between different biophysical approaches toward understanding metallobiology. We highlight three recent examples from our own laboratory. Firstly, we describe metallodrug interactions with glutathione S-transferases, an enzyme family known to attack commonly used anti-cancer drugs. We then describe a protein target for memory enhancing drugs called insulin-regulated aminopeptidase in which zinc plays a role in catalysis and regulation. Finally we describe our studies on a protein, amyloid precursor protein, that appears to play a central role in Alzheimer's disease. Copper ions have been implicated in playing both beneficial and detrimental roles in the disease by binding to different regions of this protein. PMID:22437159

  2. Simulation Studies of Protein and Small Molecule Interactions and Reaction.

    PubMed

    Yang, L; Zhang, J; Che, X; Gao, Y Q

    2016-01-01

    Computational studies of protein and small molecule (protein-ligand/enzyme-substrate) interactions become more and more important in biological science and drug discovery. Computer modeling can provide molecular details of the processes such as conformational change, binding, and transportation of small molecules/proteins, which are not easily to be captured in experiments. In this chapter, we discussed simulation studies of both protein and small molecules from three aspects: conformation sampling, transportations of small molecules in enzymes, and enzymatic reactions involving small molecules. Both methodology developments and examples of simulation studies in this field were presented. PMID:27497167

  3. Multiple protein-protein interactions converging on the Prp38 protein during activation of the human spliceosome.

    PubMed

    Schütze, Tonio; Ulrich, Alexander K C; Apelt, Luise; Will, Cindy L; Bartlick, Natascha; Seeger, Martin; Weber, Gert; Lührmann, Reinhard; Stelzl, Ulrich; Wahl, Markus C

    2016-02-01

    Spliceosomal Prp38 proteins contain a conserved amino-terminal domain, but only higher eukaryotic orthologs also harbor a carboxy-terminal RS domain, a hallmark of splicing regulatory SR proteins. We show by crystal structure analysis that the amino-terminal domain of human Prp38 is organized around three pairs of antiparallel α-helices and lacks similarities to RNA-binding domains found in canonical SR proteins. Instead, yeast two-hybrid analyses suggest that the amino-terminal domain is a versatile protein-protein interaction hub that possibly binds 12 other spliceosomal proteins, most of which are recruited at the same stage as Prp38. By quantitative, alanine surface-scanning two-hybrid screens and biochemical analyses we delineated four distinct interfaces on the Prp38 amino-terminal domain. In vitro interaction assays using recombinant proteins showed that Prp38 can bind at least two proteins simultaneously via two different interfaces. Addition of excess Prp38 amino-terminal domain to in vitro splicing assays, but not of an interaction-deficient mutant, stalled splicing at a precatalytic stage. Our results show that human Prp38 is an unusual SR protein, whose amino-terminal domain is a multi-interface protein-protein interaction platform that might organize the relative positioning of other proteins during splicing. PMID:26673105

  4. Predicting protein functions from redundancies in large-scale protein interaction networks

    NASA Technical Reports Server (NTRS)

    Samanta, Manoj Pratim; Liang, Shoudan

    2003-01-01

    Interpreting data from large-scale protein interaction experiments has been a challenging task because of the widespread presence of random false positives. Here, we present a network-based statistical algorithm that overcomes this difficulty and allows us to derive functions of unannotated proteins from large-scale interaction data. Our algorithm uses the insight that if two proteins share significantly larger number of common interaction partners than random, they have close functional associations. Analysis of publicly available data from Saccharomyces cerevisiae reveals >2,800 reliable functional associations, 29% of which involve at least one unannotated protein. By further analyzing these associations, we derive tentative functions for 81 unannotated proteins with high certainty. Our method is not overly sensitive to the false positives present in the data. Even after adding 50% randomly generated interactions to the measured data set, we are able to recover almost all (approximately 89%) of the original associations.

  5. Hepatitis E virus ORF1 encoded non structural protein-host protein interaction network.

    PubMed

    Ojha, Nishant Kumar; Lole, Kavita S

    2016-02-01

    Hepatitis E virus ORF1 encoded non-structural polyprotein (nsP) consist of multiple domains, namely: Methyltransferase, Y-domain, Protease, X-domain, Helicase and RNA dependent RNA polymerase. We have attempted to identify human liver cell proteins that are interacting with HEV ORF1 encoded functional domains by using Y2H screening. A total of 155 protein-protein interactions between HEV-ORF1 and human proteins were identified. Comparative analysis of the HEV-ORF1-Human interaction network with reconstructed human interactome showed that the cellular proteins interacting with HEV-ORF1 are central and interconnected. Enrichment analysis of Gene Ontology and cellular pathways showed that the viral proteins preferentially interacted with the proteins of metabolism and energy generation along with host immune response and ubiquitin proteasomal pathways. The mTOR and focal adhesion pathways were also targeted by the virus. These interactions suggest that HEV probably utilizes important proteins in carbohydrate metabolism, energy generation and iron homoeostasis in the host cells during its establishment. PMID:26689634

  6. Split-Cre recombinase effectively monitors protein-protein interactions in living bacteria.

    PubMed

    O'Brien, Sean P; DeLisa, Matthew P

    2014-03-01

    The ability of Cre recombinase to excise genetic material has been used extensively for genome engineering in prokaryotic and eukaryotic cells. Recently, split-Cre fragments have been described that advance control of recombinase activity in mammalian cells. However, whether these fragments can be utilized for monitoring protein-protein interactions has not been reported. In this work, we developed a protein-fragment complementation assay (PCA) based on split-Cre for monitoring and engineering pairwise protein interactions in living Escherichia coli cells. This required creation of a dual-fluorescent reporter plasmid that permits visualization of reconstituted Cre recombinase activity by switching from red to green in the presence of an interacting protein pair. The resulting split-Cre PCA faithfully links cell fluorescence with differences in binding affinity, thereby allowing the facile isolation of high-affinity binders based on phenotype. Given the resolution of its activity and sensitivity to interactions, our system may prove a viable option for poorly expressed or weakly interacting protein pairs that evade detection in other PCA formats. Based on these findings, we anticipate that our split-Cre PCA will become a highly complementary and useful new addition to the protein-protein interaction toolbox. PMID:24390935

  7. Protein-protein interactions prediction based on iterative clique extension with gene ontology filtering.

    PubMed

    Yang, Lei; Tang, Xianglong

    2014-01-01

    Cliques (maximal complete subnets) in protein-protein interaction (PPI) network are an important resource used to analyze protein complexes and functional modules. Clique-based methods of predicting PPI complement the data defection from biological experiments. However, clique-based predicting methods only depend on the topology of network. The false-positive and false-negative interactions in a network usually interfere with prediction. Therefore, we propose a method combining clique-based method of prediction and gene ontology (GO) annotations to overcome the shortcoming and improve the accuracy of predictions. According to different GO correcting rules, we generate two predicted interaction sets which guarantee the quality and quantity of predicted protein interactions. The proposed method is applied to the PPI network from the Database of Interacting Proteins (DIP) and most of the predicted interactions are verified by another biological database, BioGRID. The predicted protein interactions are appended to the original protein network, which leads to clique extension and shows the significance of biological meaning. PMID:24578640

  8. West Nile virus methyltransferase domain interacts with protein kinase G

    PubMed Central

    2013-01-01

    Background The flaviviral nonstructural protein 5 (NS5) is a phosphoprotein, though the precise identities and roles of many specific phosphorylations remain unknown. Protein kinase G (PKG), a cGMP-dependent protein kinase, has previously been shown to phosphorylate dengue virus NS5. Methods We used mass spectrometry to specifically identify NS5 phosphosites. Co-immunoprecipitation assays were used to study protein-protein interactions. Effects on viral replication were measured via replicon system and plaque assay titering. Results We identified multiple sites in West Nile virus (WNV) NS5 that are phosphorylated during a WNV infection, and showed that the N-terminal methyltransferase domain of WNV NS5 can be specifically phosphorylated by PKG in vitro. Expressing PKG in cell culture led to an enhancement of WNV viral production. We hypothesized this effect on replication could be caused by factors beyond the specific phosphorylations of NS5. Here we show for the first time that PKG is also able to stably interact with a viral substrate, WNV NS5, in cell culture and in vitro. While the mosquito-borne WNV NS5 interacted with PKG, tick-borne Langat virus NS5 did not. The methyltransferase domain of NS5 is able to mediate the interaction between NS5 and PKG, and mutating positive residues in the αE region of the methyltransferase interrupts the interaction. These same mutations completely inhibited WNV replication. Conclusions PKG is not required for WNV replication, but does make a stable interaction with NS5. While the consequence of the NS5:PKG interaction when it occurs is unclear, mutational data demonstrates that this interaction occurs in a region of NS5 that is otherwise necessary for replication. Overall, the results identify an interaction between virus and a cellular kinase and suggest a role for a host kinase in enhancing flaviviral replication. PMID:23876037

  9. Inhibition of Protein-Protein Interactions and Signaling by Small Molecules

    NASA Astrophysics Data System (ADS)

    Freire, Ernesto

    2010-03-01

    Protein-protein interactions are at the core of cell signaling pathways as well as many bacterial and viral infection processes. As such, they define critical targets for drug development against diseases such as cancer, arthritis, obesity, AIDS and many others. Until now, the clinical inhibition of protein-protein interactions and signaling has been accomplished with the use of antibodies or soluble versions of receptor molecules. Small molecule replacements of these therapeutic agents have been extremely difficult to develop; either the necessary potency has been hard to achieve or the expected biological effect has not been obtained. In this presentation, we show that a rigorous thermodynamic approach that combines differential scanning calorimetry (DSC) and isothermal titration calorimetry (ITC) provides a unique platform for the identification and optimization of small molecular weight inhibitors of protein-protein interactions. Recent advances in the development of cell entry inhibitors of HIV-1 using this approach will be discussed.

  10. Tuning protein-protein interactions using cosolvents: specific effects of ionic and non-ionic additives on protein phase behavior.

    PubMed

    Hansen, Jan; Platten, Florian; Wagner, Dana; Egelhaaf, Stefan U

    2016-04-21

    Cosolvents are routinely used to modulate the (thermal) stability of proteins and, hence, their interactions with proteins have been studied intensely. However, less is known about their specific effects on protein-protein interactions, which we characterize in terms of the protein phase behavior. We analyze the phase behavior of lysozyme solutions in the presence of sodium chloride (NaCl), guanidine hydrochloride (GuHCl), glycerol, and dimethyl sulfoxide (DMSO). We experimentally determined the crystallization boundary (XB) and, in combination with data on the cloud-point temperatures (CPTs), the crystallization gap. In agreement with other studies, our data indicate that the additives might affect the protein phase behavior through electrostatic screening and additive-specific contributions. At high salt concentrations, where electrostatic interactions are screened, both the CPT and the XB are found to be linear functions of the additive concentration. Their slopes quantify the additive-specific changes of the phase behavior and thus of the protein-protein interactions. While the specific effect of NaCl is to induce attractions between proteins, DMSO, glycerol and GuHCl (with increasing strength) weaken attractions and/or induce repulsions. Except for DMSO, changes of the CPT are stronger than those of the XB. Furthermore, the crystallization gap widens in the case of GuHCl and glycerol and narrows in the case of NaCl. We relate these changes to colloidal interaction models, namely square-well and patchy interactions. PMID:27020538

  11. Fluorous photoaffinity labeling to probe protein-small molecule interactions.

    PubMed

    Huang, Weigang; Zhang, Qisheng

    2015-01-01

    Identifying cellular targets of bioactive small molecules is essential for their applications as chemical probes or drug candidates. Of equal importance is to determine their "off-target" interactions, which usually account for unwanted properties including toxicity. Among strategies to profile small molecule-interacting proteins, photoaffinity labeling has been widely used because of its distinct advantages such as sensitivity. When combined with mass spectrometry, this approach can provide additional structural and mechanistic information, such as drug-target stoichiometry and exact interacting amino acid residues. We have described a novel fluorous photoaffinity labeling approach, in which a fluorous tag is incorporated into the photoaffinity labeling reagent to enable the enrichment of the labeled species from complex mixtures for analysis. This new feature likely makes the fluorous photoaffinity labeling approach suitable to identify transient interactions, and low-abundant, low-affinity interacting proteins in a cellular environment. PMID:25618351

  12. Co-evolutionary Analysis of Domains in Interacting Proteins Reveals Insights into Domain–Domain Interactions Mediating Protein–Protein Interactions

    PubMed Central

    Jothi, Raja; Cherukuri, Praveen F.; Tasneem, Asba; Przytycka, Teresa M.

    2006-01-01

    Recent advances in functional genomics have helped generate large-scale high-throughput protein interaction data. Such networks, though extremely valuable towards molecular level understanding of cells, do not provide any direct information about the regions (domains) in the proteins that mediate the interaction. Here, we performed co-evolutionary analysis of domains in interacting proteins in order to understand the degree of co-evolution of interacting and non-interacting domains. Using a combination of sequence and structural analysis, we analyzed protein–protein interactions in F1-ATPase, Sec23p/Sec24p, DNA-directed RNA polymerase and nuclear pore complexes, and found that interacting domain pair(s) for a given interaction exhibits higher level of co-evolution than the noninteracting domain pairs. Motivated by this finding, we developed a computational method to test the generality of the observed trend, and to predict large-scale domain–domain interactions. Given a protein–protein interaction, the proposed method predicts the domain pair(s) that is most likely to mediate the protein interaction. We applied this method on the yeast interactome to predict domain–domain interactions, and used known domain–domain interactions found in PDB crystal structures to validate our predictions. Our results show that the prediction accuracy of the proposed method is statistically significant. Comparison of our prediction results with those from two other methods reveals that only a fraction of predictions are shared by all the three methods, indicating that the proposed method can detect known interactions missed by other methods. We believe that the proposed method can be used with other methods to help identify previously unrecognized domain–domain interactions on a genome scale, and could potentially help reduce the search space for identifying interaction sites. PMID:16949097

  13. Role of Anisotropic Interactions for Proteins and Patchy Nanoparticles

    PubMed Central

    2015-01-01

    Protein–protein interactions are inherently anisotropic to some degree, with orientation-dependent interactions between repulsive and attractive or complementary regions or “patches” on adjacent proteins. In some cases it has been suggested that such patch–patch interactions dominate the thermodynamics of dilute protein solutions, as captured by the osmotic second virial coefficient (B22), but delineating when this will or will not be the case remains an open question. A series of simplified but exactly solvable models are first used to illustrate that a delicate balance exists between the strength of attractive patch–patch interactions and the patch size, and that repulsive patch–patch interactions contribute significantly to B22 for only those conditions where the repulsions are long-ranged. Finally, B22 is reformulated, without approximations, in terms of the density of states for a given interaction energy and particle–particle distance. Doing so illustrates the inherent balance of entropic and energetic contributions to B22. It highlights that simply having strong patch–patch interactions will only cause anisotropic interactions to dominate B22 solution properties if the unavoidable entropic penalties are overcome, which cannot occur if patches are too small. The results also indicate that the temperature dependence of B22 may be a simple experimental means to assess whether a small number of strongly attractive configurations dominate the dilute solution behavior. PMID:25302767

  14. Improved silicon nanowire field-effect transistors for fast protein-protein interaction screening.

    PubMed

    Lin, Ti-Yu; Li, Bor-Ran; Tsai, Sheng-Ta; Chen, Chien-Wei; Chen, Chung-Hsuan; Chen, Yit-Tsong; Pan, Chien-Yuan

    2013-02-21

    Understanding how proteins interact with each other is the basis for studying the biological mechanisms behind various physiological activities. Silicon nanowire field-effect transistors (SiNW-FETs) are sensitive sensors used to detect biomolecular interactions in real-time. However, the majority of the applications that use SiNW-FETs are for known interactions between different molecules. To explore the capability of SiNW-FETs as fast screening devices to identify unknown interacting molecules, we applied mass spectrometry (MS) to analyze molecules reversibly bound to the SiNW-FETs. Calmodulin (CaM) is a Ca(2+)-sensing protein that is ubiquitously expressed in cells and its interaction with target molecules is Ca(2+)-dependent. By modifying the SiNW-FET surface with glutathione, glutathione S-transferase (GST)-tagged CaM binds reversibly to the SiNW-FET. We first verified the Ca(2+)-dependent interaction between GST-CaM and purified troponin I, which is involved in muscle contraction, through the conductance changes of the SiNW-FET. Furthermore, the cell lysate containing overexpressed Ca(2+)/CaM-dependent protein kinase IIα induced a conductance change in the GST-CaM-modified SiNW-FET. The bound proteins were eluted and subsequently identified by MS as CaM and kinase. In another example, candidate proteins from neuronal cell lysates interacting with calneuron I (CalnI), a CaM-like protein, were captured with a GST-CalnI-modified SiNW-FET. The proteins that interacted with CalnI were eluted and verified by MS. The Ca(2+)-dependent interaction between GST-CalnI and one of the candidates, heat shock protein 70, was re-confirmed via the SiNW-FET measurement. Our results demonstrate the effectiveness of combining MS with SiNW-FETs to quickly screen interacting molecules from cell lysates. PMID:23235921

  15. Cluster conservation as a novel tool for studying protein-protein interactions evolution.

    PubMed

    Rahat, Ofer; Yitzhaky, Assif; Schreiber, Gideon

    2008-05-01

    Protein-protein interactions networks has come to be a buzzword associated with nets containing edges that represent a pair of interacting proteins (e.g. hormone-receptor, enzyme-inhibitor, antigen-antibody, and a subset of multichain biological machines). Yet, each such interaction composes its own unique network, in which vertices represent amino acid residues, and edges represent atomic contacts. Recent studies have shown that analyses of the data encapsulated in these detailed networks may impact predictions of structure-function correlation. Here, we study homologous families of protein-protein interfaces, which share the same fold but vary in sequence. In this context, we address what properties of the network are shared among relatives with different sequences (and hence different atomic interactions) and which are not. Herein, we develop the general mathematical framework needed to compare the modularity of homologous networks. We then apply this analysis to the structural data of a few interface families, including hemoglobin alpha-beta, growth hormone-receptor, and Serine protease-inhibitor. Our results suggest that interface modularity is an evolutionarily conserved property. Hence, protein-protein interfaces can be clustered down to a few modules, with the boundaries being evolutionarily conserved along homologous complexes. This suggests that protein engineering of protein-protein binding sites may be simplified by varying each module, but retaining the overall modularity of the interface. PMID:17972288

  16. Electrochemical probe for the monitoring of DNA-protein interactions.

    PubMed

    Meunier-Prest, Rita; Bouyon, Alice; Rampazzi, Eve; Raveau, Suzanne; Andreoletti, Pierre; Cherkaoui-Malki, Mustapha

    2010-08-15

    Self-assembly of thiol-terminated oligonucleotides on gold substrates provides a convenient way for DNA-functionalized surfaces. Here we describe the development of an electrochemical assay for the detection of DNA-protein interactions based on the modification of the electrochemical response of methylene blue (MB) intercalated in the DNA strands. Using a functionalized electrode with double stranded DNA carrying T3 RNA polymerase binding sequence, we show a substantial attenuation of the current upon the DNA-protein interaction. Moreover, a Langmuir binding isotherm for T3 RNA polymerase (T3 Pol) gives a dissociation constant K(D) equal to 0.46+/-0.23 microM. Such value is 100 times lower than the calculated K(D) for the non-specific interaction of bovine serum albumin (BSA) with T3 Pol promoter. In addition, the use of the T7 RNA polymerase (T7 Pol) promoter instead of the T3 Pol promoter induces an increase of K(D) from 0.46 microM to more than 25 microM. Accordingly, this strong decrease in the affinity of T3 Pol towards an off-target DNA promoter reveals an electrochemical sequence-specific discrimination of DNA-protein interactions. In conclusion, our results show that the developed electrochemical test allows the monitoring of DNA-protein interactions with high specificity and with an in situ protein detection threshold at a nanomolar range. PMID:20447818

  17. Phospho-tyrosine dependent protein–protein interaction network

    PubMed Central

    Grossmann, Arndt; Benlasfer, Nouhad; Birth, Petra; Hegele, Anna; Wachsmuth, Franziska; Apelt, Luise; Stelzl, Ulrich

    2015-01-01

    Post-translational protein modifications, such as tyrosine phosphorylation, regulate protein–protein interactions (PPIs) critical for signal processing and cellular phenotypes. We extended an established yeast two-hybrid system employing human protein kinases for the analyses of phospho-tyrosine (pY)-dependent PPIs in a direct experimental, large-scale approach. We identified 292 mostly novel pY-dependent PPIs which showed high specificity with respect to kinases and interacting proteins and validated a large fraction in co-immunoprecipitation experiments from mammalian cells. About one-sixth of the interactions are mediated by known linear sequence binding motifs while the majority of pY-PPIs are mediated by other linear epitopes or governed by alternative recognition modes. Network analysis revealed that pY-mediated recognition events are tied to a highly connected protein module dedicated to signaling and cell growth pathways related to cancer. Using binding assays, protein complementation and phenotypic readouts to characterize the pY-dependent interactions of TSPAN2 (tetraspanin 2) and GRB2 or PIK3R3 (p55γ), we exemplarily provide evidence that the two pY-dependent PPIs dictate cellular cancer phenotypes. PMID:25814554

  18. Physical Interaction between Replication Protein A (RPA) and MRN

    PubMed Central

    Oakley, Greg; Tillison, Kristin; Opiyo, Stephen; Glanzer, Jason; Horn, Jeffrey M.; Patrick, Steve M.

    2009-01-01

    Replication protein A (RPA) is a heterotrimeric protein consisting of RPA1, RPA2 and RPA3 subunits that binds to ssDNA with high affinity. The response to replication stress requires the recruitment of RPA and the MRE11/RAD50/NBS1 (MRN) complex. RPA bound to ssDNA stabilizes stalled replication forks by recruiting checkpoint proteins involved in fork stabilization. MRN can bind DNA structures encountered at stalled or collapsed replication forks, such as ssDNA-dsDNA junctions or breaks and promote the restart of DNA replication. Here, we demonstrate that RPA2 phosphorylation regulates the assembly of DNA damage-induced RPA and MRN foci. Using purified proteins, we observe a direct interaction between RPA with both NBS1 and MRE11. By utilizing RPA bound to ssDNA, we demonstrate that substituting RPA with phosphorylated RPA or a phosphomimetic decreases the interaction with the MRN complex. Also, the N-terminus of RPA1 is a critical component of the RPA-MRN protein-protein interaction. Deletion of the N-terminal oligonucleotide-oligosaccharide binding fold (OB-fold) of RPA1 abrogates RPA interactions with MRN and individual proteins of the MRN complex. Further identification of residues critical for MRN binding in the N-terminus of RPA1 show that substitution of Arg31 and Arg41 with alanines disrupts the RPA-MRN interaction and alters cell cycle progression in response to DNA damage. Thus, the N-terminus of RPA1 and phosphorylation of RPA2 regulate RPA-MRN interactions and are important in the response to DNA damage. PMID:19586055

  19. Constraints imposed by nonfunctional protein-protein interactions on gene expression and proteome size

    NASA Astrophysics Data System (ADS)

    Zhang, Jingshan; Maslov, Sergei; Shakhnovich, Eugene

    2009-03-01

    Crowded intracellular environments present a challenge for proteins to form functional specific complexes while reducing nonfunctional interactions with promiscuous nonfunctional partners. Here we show how nonfunctional interactions limit the proteome diversity and the average concentration of co-expressed and co-localized proteins. We use yeast compartments to verify our hypothesis that the yeast proteome has evolved to operate closely to the upper limit of its size, while keeping individual protein concentrations sufficiently low to reduce nonfunctional interactions. These findings have implication for conceptual understanding of intracellular compartmentalization, multicellularity, and differentiation.

  20. 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

  1. Identification of Cellular Proteins that Interact with Human Cytomegalovirus Immediate-Early Protein 1 by Protein Array Assay

    PubMed Central

    Puerta Martínez, Francisco; Tang, Qiyi

    2013-01-01

    Human cytomegalovirus (HCMV) gene expression during infection is characterized as a sequential process including immediate-early (IE), early (E), and late (L)-stage gene expression. The most abundantly expressed gene at the IE stage of infection is the major IE (MIE) gene that produces IE1 and IE2. IE1 has been the focus of study because it is an important protein, not only for viral gene expression but also for viral replication. It is believed that IE1 plays important roles in viral gene regulation by interacting with cellular proteins. In the current study, we performed protein array assays and identified 83 cellular proteins that interact with IE1. Among them, seven are RNA-binding proteins that are important in RNA processing; more than half are nuclear proteins that are involved in gene regulations. Tumorigenesis-related proteins are also found to interact with IE1, implying that the role of IE1 in tumorigenesis might need to be reevaluated. Unexpectedly, cytoplasmic proteins, such as Golgi autoantigen and GGA1 (both related to the Golgi trafficking protein), are also found to be associated with IE1. We also employed a coimmunoprecipitation assay to test the interactions of IE1 and some of the proteins identified in the protein array assays and confirmed that the results from the protein array assays are reliable. Many of the proteins identified by the protein array assay have not been previously reported. Therefore, the functions of the IE1-protein interactions need to be further explored in the future. PMID:24385082

  2. Electrostatic Interactions in the Denatured State Ensemble: Their Effect Upon Protein Folding and Protein Stability

    PubMed Central

    Sato, Satoshi; Horng, Jia-Cherng; Anil, Burcu

    2009-01-01

    It is now recognized that the denatured state ensemble (DSE) of proteins can contain significant amounts of structure, particularly under native conditions. Well-studied examples include small units of hydrogen bonded secondary structure, particularly helices or turns as well hydrophobic clusters. Other types of interactions are less well characterized and it has often been assumed that electrostatic interactions play at most a minor role in the DSE. However, recent studies have shown that both favorable and unfavorable electrostatic interactions can be formed in the DSE. These can include surprisingly specific non-native interactions that can even persist in the transition state for protein folding. DSE electrostatic interactions can be energetically significant and their modulation either by mutation or by varying solution conditions can have a major impact upon protein stability. pH dependent stability studies have shown that electrostatic interactions can contribute up to 4 kcal mol−1 to the stability of the DSE. PMID:17900519

  3. Specific ion and buffer effects on protein-protein interactions of a monoclonal antibody.

    PubMed

    Roberts, D; Keeling, R; Tracka, M; van der Walle, C F; Uddin, S; Warwicker, J; Curtis, R

    2015-01-01

    Better predictive ability of salt and buffer effects on protein-protein interactions requires separating out contributions due to ionic screening, protein charge neutralization by ion binding, and salting-in(out) behavior. We have carried out a systematic study by measuring protein-protein interactions for a monoclonal antibody over an ionic strength range of 25 to 525 mM at 4 pH values (5, 6.5, 8, and 9) in solutions containing sodium chloride, calcium chloride, sodium sulfate, or sodium thiocyante. The salt ions are chosen so as to represent a range of affinities for protein charged and noncharged groups. The results are compared to effects of various buffers including acetate, citrate, phosphate, histidine, succinate, or tris. In low ionic strength solutions, anion binding affinity is reflected by the ability to reduce protein-protein repulsion, which follows the order thiocyanate > sulfate > chloride. The sulfate specific effect is screened at the same ionic strength required to screen the pH dependence of protein-protein interactions indicating sulfate binding only neutralizes protein charged groups. Thiocyanate specific effects occur over a larger ionic strength range reflecting adsorption to charged and noncharged regions of the protein. The latter leads to salting-in behavior and, at low pH, a nonmonotonic interaction profile with respect to sodium thiocyanate concentration. The effects of thiocyanate can not be rationalized in terms of only neutralizing double layer forces indicating the presence of an additional short-ranged protein-protein attraction at moderate ionic strength. Conversely, buffer specific effects can be explained through a charge neutralization mechanism, where buffers with greater valency are more effective at reducing double layer forces at low pH. Citrate binding at pH 6.5 leads to protein charge inversion and the formation of attractive electrostatic interactions. Throughout the report, we highlight similarities in the measured

  4. Modeling of metal interaction geometries for protein-ligand docking.

    PubMed

    Seebeck, Birte; Reulecke, Ingo; Kämper, Andreas; Rarey, Matthias

    2008-05-15

    The accurate modeling of metal coordination geometries plays an important role for structure-based drug design applied to metalloenzymes. For the development of a new metal interaction model, we perform a statistical analysis of metal interaction geometries that are relevant to protein-ligand complexes. A total of 43,061 metal sites of the Protein Data Bank (PDB), containing amongst others magnesium, calcium, zinc, iron, manganese, copper, cadmium, cobalt, and nickel, were evaluated according to their metal coordination geometry. Based on statistical analysis, we derived a model for the automatic calculation and definition of metal interaction geometries for the purpose of molecular docking analyses. It includes the identification of the metal-coordinating ligands, the calculation of the coordination geometry and the superposition of ideal polyhedra to identify the optimal positions for free coordination sites. The new interaction model was integrated in the docking software FlexX and evaluated on a data set of 103 metalloprotein-ligand complexes, which were extracted from the PDB. In a first step, the quality of the automatic calculation of the metal coordination geometry was analyzed. In 74% of the cases, the correct prediction of the coordination geometry could be determined on the basis of the protein structure alone. Secondly, the new metal interaction model was tested in terms of predicting protein-ligand complexes. In the majority of test cases, the new interaction model resulted in an improved docking accuracy of the top ranking placements. PMID:18041759

  5. Redesign of a Protein-peptide Interaction: Characterization and Applications

    SciTech Connect

    Jackrel, M.; Valverde, R; Regan, L

    2009-01-01

    The design of protein-peptide interactions has a wide array of practical applications and also reveals insight into the basis for molecular recognition. Here, we present the redesign of a tetratricopeptide repeat (TPR) protein scaffold, along with its corresponding peptide ligand. We show that the binding properties of these protein-peptide pairs can be understood, quantitatively, using straightforward chemical considerations. The recognition pairs we have developed are also practically useful for the specific identification of tagged proteins. We demonstrate the facile replacement of these proteins, which we have termed T-Mods (TPR-based recognition module), for antibodies in both detection and purification applications. The new protein-peptide pair has a dissociation constant that is weaker than typical antibody-antigen interactions, yet the recognition pair is highly specific and we have shown that this affinity is sufficient for both Western blotting and affinity purification. Moreover, we demonstrate that this more moderate affinity is actually advantageous for purification applications, because extremely harsh conditions are not required to dissociate the T-Mod-peptide interaction. The results we present are important, not only because they represent a successful application of protein design but also because they help define the properties that should be sought in other scaffolds that are being developed as antibody replacements.

  6. Machine Learning of Protein Interactions in Fungal Secretory Pathways.

    PubMed

    Kludas, Jana; Arvas, Mikko; Castillo, Sandra; Pakula, Tiina; Oja, Merja; Brouard, Céline; Jäntti, Jussi; Penttilä, Merja; Rousu, Juho

    2016-01-01

    In this paper we apply machine learning methods for predicting protein interactions in fungal secretion pathways. We assume an inter-species transfer setting, where training data is obtained from a single species and the objective is to predict protein interactions in other, related species. In our methodology, we combine several state of the art machine learning approaches, namely, multiple kernel learning (MKL), pairwise kernels and kernelized structured output prediction in the supervised graph inference framework. For MKL, we apply recently proposed centered kernel alignment and p-norm path following approaches to integrate several feature sets describing the proteins, demonstrating improved performance. For graph inference, we apply input-output kernel regression (IOKR) in supervised and semi-supervised modes as well as output kernel trees (OK3). In our experiments simulating increasing genetic distance, Input-Output Kernel Regression proved to be the most robust prediction approach. We also show that the MKL approaches improve the predictions compared to uniform combination of the kernels. We evaluate the methods on the task of predicting protein-protein-interactions in the secretion pathways in fungi, S.cerevisiae, baker's yeast, being the source, T. reesei being the target of the inter-species transfer learning. We identify completely novel candidate secretion proteins conserved in filamentous fungi. These proteins could contribute to their unique secretion capabilities. PMID:27441920

  7. Machine Learning of Protein Interactions in Fungal Secretory Pathways

    PubMed Central

    Kludas, Jana; Arvas, Mikko; Castillo, Sandra; Pakula, Tiina; Oja, Merja; Brouard, Céline; Jäntti, Jussi; Penttilä, Merja

    2016-01-01

    In this paper we apply machine learning methods for predicting protein interactions in fungal secretion pathways. We assume an inter-species transfer setting, where training data is obtained from a single species and the objective is to predict protein interactions in other, related species. In our methodology, we combine several state of the art machine learning approaches, namely, multiple kernel learning (MKL), pairwise kernels and kernelized structured output prediction in the supervised graph inference framework. For MKL, we apply recently proposed centered kernel alignment and p-norm path following approaches to integrate several feature sets describing the proteins, demonstrating improved performance. For graph inference, we apply input-output kernel regression (IOKR) in supervised and semi-supervised modes as well as output kernel trees (OK3). In our experiments simulating increasing genetic distance, Input-Output Kernel Regression proved to be the most robust prediction approach. We also show that the MKL approaches improve the predictions compared to uniform combination of the kernels. We evaluate the methods on the task of predicting protein-protein-interactions in the secretion pathways in fungi, S.cerevisiae, baker’s yeast, being the source, T. reesei being the target of the inter-species transfer learning. We identify completely novel candidate secretion proteins conserved in filamentous fungi. These proteins could contribute to their unique secretion capabilities. PMID:27441920

  8. Interactions between Phage-Shock Proteins in Escherichia coli

    PubMed Central

    Adams, Hendrik; Teertstra, Wieke; Demmers, Jeroen; Boesten, Rolf; Tommassen, Jan

    2003-01-01

    Expression of the pspABCDE operon of Escherichia coli is induced upon infection by filamentous phage and by many other stress conditions, including defects in protein export. Expression of the operon requires the alternative sigma factor σ54 and the transcriptional activator PspF. In addition, PspA plays a negative regulatory role, and the integral-membrane proteins PspB and PspC play a positive one. In this study, we investigated whether the suggested protein-protein interactions implicated in this complex regulatory network can indeed be demonstrated. Antisera were raised against PspB, PspC, and PspD, which revealed, in Western blotting experiments, that PspC forms stable sodium dodecyl sulfate-resistant dimers and that the hypothetical pspD gene is indeed expressed in vivo. Fractionation experiments showed that PspD localizes as a peripherally bound inner membrane protein. Cross-linking studies with intact cells revealed specific interactions of PspA with PspB and PspC, but not with PspD. Furthermore, affinity-chromatography suggested that PspB could bind PspA only in the presence of PspC. These data indicate that regulation of the psp operon is mediated via protein-protein interactions. PMID:12562786

  9. A residue level protein-protein interaction model in electrolyte solutions

    NASA Astrophysics Data System (ADS)

    Song, Xueyu

    2014-03-01

    The osmotic second virial coefficients B2 are directly related to the solubility of protein molecules in electrolyte solutions and can be useful to narrow down the search parameter space of protein crystallization conditions. Using a residue level model of protein-protein interaction in electrolyte solutions B2 of bovine pancreatic trypsin inhibitor and lysozyme in various solution conditions such as salt concentration, pH and temperature are calculated using an extended Fast Multipole Methods in combination with the boundary element formulation. Overall, the calculated B2 are well correlated with the experimental observations for various solution conditions. In combination with our previous work on the binding affinity calculations of protein complexes it is demonstrated that our residue level model can be used as a reliable model to describe protein-protein interaction in solutions.

  10. Protein/Protein Interactions in the Mammalian Heme Degradation Pathway

    PubMed Central

    Spencer, Andrea L. M.; Bagai, Ireena; Becker, Donald F.; Zuiderweg, Erik R. P.; Ragsdale, Stephen W.

    2014-01-01

    Heme oxygenase (HO) catalyzes the rate-limiting step in the O2-dependent degradation of heme to biliverdin, CO, and iron with electrons delivered from NADPH via cytochrome P450 reductase (CPR). Biliverdin reductase (BVR) then catalyzes conversion of biliverdin to bilirubin. We describe mutagenesis combined with kinetic, spectroscopic (fluorescence and NMR), surface plasmon resonance, cross-linking, gel filtration, and analytical ultracentrifugation studies aimed at evaluating interactions of HO-2 with CPR and BVR. Based on these results, we propose a model in which HO-2 and CPR form a dynamic ensemble of complex(es) that precede formation of the productive electron transfer complex. The 1H-15N TROSY NMR spectrum of HO-2 reveals specific residues, including Leu-201, near the heme face of HO-2 that are affected by the addition of CPR, implicating these residues at the HO/CPR interface. Alanine substitutions at HO-2 residues Leu-201 and Lys-169 cause a respective 3- and 22-fold increase in Km values for CPR, consistent with a role for these residues in CPR binding. Sedimentation velocity experiments confirm the transient nature of the HO-2·CPR complex (Kd = 15.1 μm). Our results also indicate that HO-2 and BVR form a very weak complex that is only captured by cross-linking. For example, under conditions where CPR affects the 1H-15N TROSY NMR spectrum of HO-2, BVR has no effect. Fluorescence quenching experiments also suggest that BVR binds HO-2 weakly, if at all, and that the previously reported high affinity of BVR for HO is artifactual, resulting from the effects of free heme (dissociated from HO) on BVR fluorescence. PMID:25196843

  11. Dynamics and mechanism of ultrafast water–protein interactions

    PubMed Central

    Qin, Yangzhong; Wang, Lijuan; Zhong, Dongping

    2016-01-01

    Protein hydration is essential to its structure, dynamics, and function, but water–protein interactions have not been directly observed in real time at physiological temperature to our awareness. By using a tryptophan scan with femtosecond spectroscopy, we simultaneously measured the hydration water dynamics and protein side-chain motions with temperature dependence. We observed the heterogeneous hydration dynamics around the global protein surface with two types of coupled motions, collective water/side-chain reorientation in a few picoseconds and cooperative water/side-chain restructuring in tens of picoseconds. The ultrafast dynamics in hundreds of femtoseconds is from the outer-layer, bulk-type mobile water molecules in the hydration shell. We also found that the hydration water dynamics are always faster than protein side-chain relaxations but with the same energy barriers, indicating hydration shell fluctuations driving protein side-chain motions on the picosecond time scales and thus elucidating their ultimate relationship. PMID:27339138

  12. Dynamics and mechanism of ultrafast water-protein interactions.

    PubMed

    Qin, Yangzhong; Wang, Lijuan; Zhong, Dongping

    2016-07-26

    Protein hydration is essential to its structure, dynamics, and function, but water-protein interactions have not been directly observed in real time at physiological temperature to our awareness. By using a tryptophan scan with femtosecond spectroscopy, we simultaneously measured the hydration water dynamics and protein side-chain motions with temperature dependence. We observed the heterogeneous hydration dynamics around the global protein surface with two types of coupled motions, collective water/side-chain reorientation in a few picoseconds and cooperative water/side-chain restructuring in tens of picoseconds. The ultrafast dynamics in hundreds of femtoseconds is from the outer-layer, bulk-type mobile water molecules in the hydration shell. We also found that the hydration water dynamics are always faster than protein side-chain relaxations but with the same energy barriers, indicating hydration shell fluctuations driving protein side-chain motions on the picosecond time scales and thus elucidating their ultimate relationship. PMID:27339138

  13. Computational prediction of virus-human protein-protein interactions using embedding kernelized heterogeneous data.

    PubMed

    Nourani, Esmaeil; Khunjush, Farshad; Durmuş, Saliha

    2016-05-24

    Pathogenic microorganisms exploit host cellular mechanisms and evade host defense mechanisms through molecular pathogen-host interactions (PHIs). Therefore, comprehensive analysis of these PHI networks should be an initial step for developing effective therapeutics against infectious diseases. Computational prediction of PHI data is gaining increasing demand because of scarcity of experimental data. Prediction of protein-protein interactions (PPIs) within PHI systems can be formulated as a classification problem, which requires the knowledge of non-interacting protein pairs. This is a restricting requirement since we lack datasets that report non-interacting protein pairs. In this study, we formulated the "computational prediction of PHI data" problem using kernel embedding of heterogeneous data. This eliminates the abovementioned requirement and enables us to predict new interactions without randomly labeling protein pairs as non-interacting. Domain-domain associations are used to filter the predicted results leading to 175 novel PHIs between 170 human proteins and 105 viral proteins. To compare our results with the state-of-the-art studies that use a binary classification formulation, we modified our settings to consider the same formulation. Detailed evaluations are conducted and our results provide more than 10 percent improvements for accuracy and AUC (area under the receiving operating curve) results in comparison with state-of-the-art methods. PMID:27072625

  14. Evolution and protein interactions of AP2 proteins in Brassicaceae: Evidence linking development and environmental responses.

    PubMed

    Zeng, Liping; Yin, Yue; You, Chenjiang; Pan, Qianli; Xu, Duo; Jin, Taijie; Zhang, Bailong; Ma, Hong

    2016-06-01

    Plants have evolved a large number of transcription factors (TF), which are enriched among duplicate genes, highlighting their roles in complex regulatory networks. The APETALA2/EREBP-like genes constitute a large plant TF family and participate in development and stress responses. To probe the conservation and divergence of AP2/EREBP genes, we analyzed the duplication patterns of this family in Brassicaceae and identified interacting proteins of representative Arabidopsis AP2/EREBP proteins. We found that many AP2/EREBP duplicates generated early in Brassicaceae history were quickly lost, but many others were retained in all tested Brassicaceae species, suggesting early functional divergence followed by persistent conservation. In addition, the sequences of the AP2 domain and exon numbers were highly conserved in rosids. Furthermore, we used 16 A. thaliana AP2/EREBP proteins as baits in yeast screens and identified 1,970 potential AP2/EREBP-interacting proteins, with a small subset of interactions verified in planta. Many AP2 genes also exhibit reduced expression in an anther-defective mutant, providing a possible link to developmental regulation. The putative AP2-interacting proteins participate in many functions in development and stress responses, including photomorphogenesis, flower development, pathogenesis, drought and cold responses, abscisic acid and auxin signaling. Our results present the AP2/EREBP evolution patterns in Brassicaceae, and support a proposed interaction network of AP2/EREBP proteins and their putative interacting proteins for further study. PMID:26472270

  15. 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 pr