Visualization of unfavorable interactions in protein folds

Summary: Three dimensional structures of proteins contain errorswhich often originate from limitations of the experimental techniquesemployed. Such errors frequently result in unfavorable atomicinteractions. Here we present a new web service, called InteractionViewer, for the visualization and correction of such errors.We show how the Interaction Viewer is used in combination withthe NQ-Flipper service to spot strained asparagine and glutaminerotamers and we emphasize the convenience of this service incorrecting such errors. Availability: The web service is integrated with the NQ-Flipperservice and accessible at http://flipper.services.came.sbg.ac.at Contact: sippl{at}came.sbg.ac.at Associate Editor: Anna Tramontano     

Visualization of protein interactions in living Caenorhabditis elegans using bimolecular fluorescence complementation analysis

The bimolecular fluorescence complementation (BiFC) assay is a powerful tool for visualizing and identifying protein interactions in living cells. This assay is based on the principle of protein-fragment complementation, using two nonfluorescent fragments derived from fluorescent proteins. When two fragments are brought together in living cells by tethering each to one of a pair of interacting proteins, fluorescence is restored. Here, we provide a protocol for a Venus-based BiFC assay to visualize protein interactions in the living nematode, Caenorhabditis elegans. We discuss how to design appropriate C. elegans BiFC cloning vectors to enable visualization of protein interactions using either inducible heat shock promoters or native promoters; transform the constructs into worms by microinjection; and analyze and interpret the resulting data. When expression of BiFC fusion proteins is induced by heat shock, the fluorescent signals can be visualized as early as 30 min after induction and last for 24 h in transgenic animals. The entire procedure takes 2-3 weeks to complete.     

Visualization and characterization of RNA–protein interactions in living cells

RNA–protein interactions are the structural and functional basis of significant numbers of RNA molecules. RNA–protein interaction assays though, still mainly depend on biochemical tests in vitro. Here, we establish a convenient and reliable RNA fluorescent three-hybrid (rF3H) method to detect/interrogate the interactions between RNAs and proteins in cells. A GFP tagged highly specific RNA trap is constructed to anchor the RNA of interest to an artificial or natural subcellular structure, and RNA–protein interactions can be detected and visualized by the enrichment of RNA binding proteins (RBPs) at these structures. Different RNA trapping systems are developed and detection of RNA–protein complexes at multiple subcellular structures are assayed. With this new toolset, interactions between proteins and mRNA or noncoding RNAs are characterized, including the interaction between a long noncoding RNA and an epigenetic modulator. Our approach provides a flexible and reliable method for the characterization of RNA–protein interactions in living cells.     

NMR analysis of protein interactions

Recent technological advances in NMR spectroscopy have alleviated the size limitations for the determination of biomolecular structures in solution. At the same time, novel NMR parameters such as residual dipolar couplings are providing greater accuracy. As this review shows, the structures of protein-protein and protein-nucleic acid complexes up to 50 kDa can now be accurately determined. Although de novo structure determination still requires considerable effort, information on interaction surfaces from chemical shift perturbations is much easier to obtain. Advances in modelling and data-driven docking procedures allow this information to be used for determining approximate structures of biomolecular complexes. As a result, a wealth of information has become available on the way in which proteins interact with other biomolecules. Of particular interest is the fact that these NMR-based methods can be applied to weak and transient protein-protein complexes that are difficult to study by other structural methods.     

Mass spectrometric analysis of protein interactions

Mass spectrometry is a powerful tool for identification of interaction partners and structural characterization of protein interactions because of its high sensitivity, mass accuracy and tolerance towards sample heterogeneity. Several tools that allow studies of protein interaction are now available and recent developments that increase the confidence of studies of protein interaction by mass spectrometry include quantification of affinity-purified proteins by stable isotope labeling and reagents for surface topology studies that can be identified by mass-contributing reporters (e.g. isotope labels, cleavable cross-linkers or fragment ions. The use of mass spectrometers to study protein interactions using deuterium exchange and for analysis of intact protein complexes recently has progressed considerably.     

PIANA: protein interactions and network analysis

We present a software framework and tool called Protein Interactions And Network Analysis (PIANA) that facilitates working with protein interaction networks by (1) integrating data from multiple sources, (2) providing a library that handles graph-related tasks and (3) automating the analysis of protein-protein interaction networks. PIANA can also be used as a stand-alone application to create protein interaction networks and perform tasks such as predicting protein interactions and helping to identify spots in a 2D electrophoresis gel. Availability: PIANA is under the GNU GPL. Source code, database and detailed documentation may be freely downloaded from http://sbi.imim.es/piana.     

Computational structural analysis of protein interactions and networks

Protein interactions have been at the focus of computational biology in recent years. In particular, interest has come from two different communities--structural and systems biology. Here, we will discuss key systems and structural biology methods that have been used for analysis and prediction of protein-protein interactions and the insight these approaches have provided on the nature and organization of protein-protein interactions inside cells.     

Proteomics analysis identifies phosphorylation-dependent alpha-synuclein protein interactions

Mutations and copy number variation in the SNCA gene encoding the neuronal protein alpha-synuclein have been linked to familial Parkinson disease (Thomas, B., and Beal, M. F. (2007) Parkinson's disease. Hum. Mol. Genet. 16, R183-R194). The carboxyl terminus of alpha-synuclein can be phosphorylated at tyrosine 125 and serine 129, although only a small fraction of the protein is phosphorylated under normal conditions (Okochi, M., Walter, J., Koyama, A., Nakajo, S., Baba, M., Iwatsubo, T., Meijer, L., Kahle, P. J., and Haass, C. (2000) Constitutive phosphorylation of the Parkinson's disease associated alpha-synuclein. J. Biol. Chem. 275, 390-397). Under pathological conditions, such as in Parkinson disease, alpha-synuclein is a major component of Lewy bodies, a pathological hallmark of Parkinson disease, and is mostly phosphorylated at Ser-129 (Anderson, J. P., Walker, D. E., Goldstein, J. M., de Laat, R., Banducci, K., Caccavello, R. J., Barbour, R., Huang, J. P., Kling, K., Lee, M., Diep, L., Keim, P. S., Shen, X. F., Chataway, T., Schlossmacher, M. G., Seubert, P., Schenk, D., Sinha, S., Gai, W. P., and Chilcote, T. J. (2006) Phosphorylation of Ser-129 is the dominant pathological modification of alpha-synuclein in familial and sporadic Lewy body disease. J. Biol. Chem. 281, 29739-29752). Controversy exists over the extent to which phosphorylation of alpha-synuclein and/or the visible protein aggregation in Lewy bodies are steps in disease pathogenesis, are protective, or are neutral markers for the disease process. Here we used the combination of peptide pulldown assays and mass spectrometry to identify and compare protein-protein interactions of phosphorylated and non-phosphorylated alpha-synuclein. We showed that non-phosphorylated alpha-synuclein carboxyl terminus pulled down protein complexes that were highly enriched for mitochondrial electron transport proteins, whereas alpha-synuclein carboxyl terminus phosphorylated on either Ser-129 or Tyr-125 did not. Instead the set of proteins pulled down by phosphorylated alpha-synuclein was highly enriched in certain cytoskeletal proteins, in vesicular trafficking proteins, and in a small number of enzymes involved in protein serine phosphorylation. This targeted comparative proteomics approach for unbiased identification of protein-protein interactions suggests that there are functional consequences when alpha-synuclein is phosphorylated.     

Thermodynamic analysis of protein interactions with biosensor technology

A methodology using biosensor technology for combined kinetic and thermodynamic analysis of biomolecular interactions is described. Rate and affinity constants are determined with BIAcore. Thermodynamics parameters, changes in free energy, enthalpy and entropy, are evaluated from equilibrium data and by using rate constants and transition state theory. The methodology using van't Hoff theory gives complementary information to microcalorimetry, since only the direct binding is measured with BIAcore whereas microcalorimetry measures all components, including e.g. hydration effects. Furthermore, BIAcore gives possibilities to gain new information by thermodynamic analysis of the rate constants.     

Visualization of molecular interactions by fluorescence complementation

The visualization of protein complexes in living cells enables the examination of protein interactions in their normal environment and the determination of their subcellular localization. The bimolecular fluorescence complementation assay has been used to visualize interactions among multiple proteins in many cell types and organisms. Modified forms of this assay have been used to visualize the competition between alternative interaction partners and the covalent modification of proteins by ubiquitin-family peptides.     

Protein recruitment systems for the analysis of protein +/- protein interactions

The yeast Saccharomyces cerevisiae serves as an excellent genetic tool for the analysis of protein +/- protein interactions. The most common system, used to date, is the two-hybrid system. Although proven very powerful, the two-hybrid system exhibits several inherent problems and limitations. Recently, two alternative systems have been described that take advantage of the fact that localization of signal transduction effectors to the inner leaflet of the plasma membrane is absolutely necessary for yeast viability. These effectors can either be the Ras guanyl nucleotide exchange factor or Ras itself. The yeast strain used in both systems is a temperature-sensitive mutant in the yeast Ras guanyl nucleotide exchange factor, CDC25. Membrane localization of these effectors is achieved via protein +/- protein interaction. Each system can be used to test interaction between known protein pairs, as well as for isolation of novel protein interactions. Described here are the scientific and technical steps to be considered for both protein recruitment systems.     

Fluorescence analysis of receptor-G protein interactions in cell membranes

The dynamics of G protein heterotrimer complex formation and disassembly in response to nucleotide binding and receptor activation govern the rate of responses to external stimuli. We use a novel flow cytometry approach to study the effects of lipid modification, isoform specificity, lipid environment, and receptor stimulation on the affinity and kinetics of G protein subunit binding. Fluorescein-labeled myristoylated Galpha(i1) (F-alpha(i1)) was used as the ligand bound to Gbetagamma in competition binding studies with differently modified Galpha subunit isoforms. In detergent solutions, the binding affinity of Galpha(i) to betagamma was 2 orders of magnitude higher than for Galpha(o) and Galpha(s) (IC50 of 0.2 nM vs 17 and 27 nM, respectively), while in reconstituted bovine brain lipid vesicles, binding was slightly weaker. The effects of receptor on the G protein complex were assessed in alpha(2A)AR receptor expressing CHO cell membranes into which purified betagamma subunits and F-alpha(i1) were reconstituted. These cell membrane studies led to the following observations: (1) binding of alpha subunit to the betagamma was not enhanced by receptor in the presence or absence of agonist, indicating that betagamma contributed essentially all of the binding energy for alpha(i1) interaction with the membrane; (2) activation of the receptor facilitated GTPgammaS-stimulated detachment of F-alpha(i1) from betagamma and the membrane. Thus flow cytometry permits quantiatitive and real-time assessments of protein-protein interactions in complex membrane environments.     

Flow cytometric analysis of immunoprecipitates: high-throughput analysis of protein phosphorylation and protein-protein interactions

BACKGROUND: Activation-induced protein phosphorylation can be studied by Western blotting, but this method is time consuming and depends on the use of radioactive probes for quantitation. We present a novel assay for the assessment of protein phosphorylation based on latex particles and flow cytometry. METHODS: This method employs monoclonal antibodies coupled to latex particles to immobilize protein kinase substrates. Their phosphorylation status is assessed by reactivity with phosphoepitope-specific antibodies. The amount of immobilized protein on the particles was analyzed by direct or indirect immunofluorescence with antibodies to nonphosphorylated epitopes. RESULTS: The assay allowed measurement of phosphorylation of multiple protein kinase substrates in stimulated T cells, including the zeta chain of the T-cell receptor, ZAP-70, CD3, CD5, SHP-1, and ERK-2, using 1-3 microg of total cell protein per sample. The assay provided high resolution of kinetics of phosphorylation and dephosphorylation. Interactions of protein kinase substrates with associated signaling molecules were demonstrated. CONCLUSIONS: The novel assay allows high-throughput quantitative measurement of protein modifications during signal transduction.     

Visualization and sensing of intermolecular interactions with two-color fluorescent probes

We developed a new generic fluorescence sensing technology based on the change of relative intensities between two well-separated emission bands of the novel functional 3-hydroxychromone (3HC) dyes. A greatly enhanced self-calibrating wavelength-ratiometric response is obtained to all major types of non-covalent interactions that can be used in sensing--to polarity, hydrogen bonding ability and to local electrostatic fields. This technology may find a broad range of applications--from homogeneous assays in solutions to microarrays, microfluidic devices, nanosensors and whole cell imaging systems. It allows transforming micelles or phospholipid vesicles into nanosensor devices. In cellular research a high sensitivity to membrane potentials can be obtained and the membrane changes during apoptosis detected.     

Network representation of protein interactions: Theory of graph description and analysis

A methodological framework is presented for the graph theoretical interpretation of NMR data of protein interactions. The proposed analysis generalizes the idea of network representations of protein structures by expanding it to protein interactions. This approach is based on regularization of residue‐resolved NMR relaxation times and chemical shift data and subsequent construction of an adjacency matrix that represents the underlying protein interaction as a graph or network. The network nodes represent protein residues. Two nodes are connected if two residues are functionally correlated during the protein interaction event. The analysis of the resulting network enables the quantification of the importance of each amino acid of a protein for its interactions. Furthermore, the determination of the pattern of correlations between residues yields insights into the functional architecture of an interaction. This is of special interest for intrinsically disordered proteins, since the structural (three‐dimensional) architecture of these proteins and their complexes is difficult to determine. The power of the proposed methodology is demonstrated at the example of the interaction between the intrinsically disordered protein osteopontin and its natural ligand heparin.     

Investigation of Fanconi anemia protein interactions by yeast two-hybrid analysis

Fanconi anemia is a chromosomal breakage disorder with eight complementation groups (A-H), and three genes (FANCA, FANCC, and FANCG) have been identified. Initial investigations of the interaction between FANCA and FANCC, principally by co-immunoprecipitation, have proved controversial. We used the yeast two-hybrid assay to test for interactions of the FANCA, FANCC, and FANCG proteins. No activation of the reporter gene was observed in yeast co-expressing FANCA and FANCC as hybrid proteins, suggesting that FANCA does not directly interact with FANCC. However, a high level of activation was found when FANCA was co-expressed with FANCG, indicating strong, direct interaction between these proteins. Both FANCA and FANCG show weak but consistent interaction with themselves, suggesting that their function may involve dimerisation. The site of interaction of FANCG with FANCA was investigated by analysis of 12 mutant fragments of FANCG. Although both N- and C-terminal fragments did interact, binding to FANCA was drastically reduced, suggesting that more than one region of the FANCG protein is required for proper interaction with FANCA.     

Quantitative analysis of cohesin complex stoichiometry and SMC3 modification-dependent protein interactions

Cohesin is a protein complex that plays an essential role in pairing replicated sister chromatids during cell division. The vertebrate cohesin complex consists of four core components including structure maintenance of chromosomes proteins SMC1 and SMC3, RAD21, and SA2/SA1. Extensive research suggests that cohesin traps the sister chromatids by a V-shaped SMC1/SMC3 heterodimer bound to the RAD21 protein that closes the ring. Accordingly, the single "ring" model proposes that two sister chromatids are trapped in a single ring that is composed of one molecule each of the 4 subunits. However, evidence also exists for alternative models. The hand-cuff model suggests that each sister chromatid is trapped individually by two rings that are joined through the shared SA1/SA2 subunit. We report here the determination of cohesin subunit stoichiometry of endogenous cohesin complex by quantitative mass spectrometry. Using qConCAT-based isotope labeling, we show that the cohesin core complex contains equimolar of the 4 core components, suggesting that each cohesin ring is closed by one SA1/SA2 molecule. Furthermore, we applied this strategy to quantify post-translational modification-dependent cohesin interactions. We demonstrate that quantitative mass spectrometry is a powerful tool for measuring stoichiometry of endogenous protein core complex.