Protein-protein interactions refer to the association of
proteinmolecules and the study of these associations from the perspective of biochemistry, signal transductionand networks.
The interactions between proteins are important for many biological functions. For example, signals from the exterior of a cell are mediated to the inside of that cell by protein-protein interactions of the signaling molecules. This process, called
signal transduction, plays a fundamental role in many biological processes and in many diseases (e.g. cancer). Proteins might interact for a long time to form part of a protein complex, a protein may be carrying another protein (for example, from cytoplasmto nucleus or vice versa in the case of the nuclear poreimportins), or a protein may interact briefly with another protein just to modify it (for example, a protein kinasewill add a phosphate to a target protein). This modification of proteins can itself change protein-protein interactions. For example, some proteins with SH2 domains only bind to other proteins when they are phosphorylated on the amino acidtyrosine while bromodomainsspecifically recognise acetylated lysines. In conclusion, protein-protein interactions are of central importance for virtually every process in a living cell. Information about these interactions improves our understanding of diseases and can provide the basis for new therapeutic approaches.
Methods to investigate protein-protein interactions
As protein-protein interactions are so important there are a multitude of methods to detect them. Each of the approaches has its own strengths and weaknesses, especially with regard to the
sensitivityand specificityof the method. A high sensitivity means that many of the interactions that occur in reality are detected by the screen. A high specificity indicates that most of the interactions detected by the screen are also occurring in reality.
Co-immunoprecipitationis considered to be the gold standard assay for protein-protein interactions, especially when it is performed with endogenous (not overexpressed and not tagged) proteins. The protein of interest is isolated with a specific antibody. Interaction partners which stick to this protein are subsequently identified by western blotting. Interactions detected by this approach are considered to be real. However, this method can only verify interactions between suspected interaction partners. Thus, it is not a screening approach.
Bimolecular Fluorescence Complementation(BiFC) is a new technique in observing the interactions of proteins. Combining with other new techniques, this method can be used to screen protein-protein interactions and their modulators [cite journal| author=Lu JP, Beatty LK, Pinthus JH. |date=2008 |title=Dual expression recombinase based (DERB) single vector system for high throughput screening and verification of protein interactions in living cells.|journal=Nature Precedings
Fluorescence resonance energy transfer(FRET) is a common technique when observing the interactions of only two different proteins.
*Pull-down assays are a common variation of immunoprecipitation and are used identically, although this approach is more amenable to an initial screen for interacting proteins.
Label transfercan be used for screening or confirmation of protein interactions and can provide information about the interface where the interaction takes place. Label transfer can also detect weak or transient interactions that are difficult to capture using other "in vitro" detection strategies. In a label transfer reaction, a known protein is tagged with a detectable label. The label is then passed to an interacting protein, which can then be identified by the presence of the label.
yeast two-hybridscreen investigates the interaction between artificial fusion proteins inside the nucleus of yeast. This approach can identify binding partners of a protein in an unbiased manner. However, the method has a notorious high false-positive rate which makes it necessary to verify the identified interactions by co-immunoprecipitation.
*"In-vivo" crosslinking of protein complexes using
photo-reactive amino acid analogs was introduced in 2005 by researchers from the Max Planck Institute [cite journal| author=Suchanek, M., Radzikowska, A., and Thiele, C. |date=2005 |title=Photo-leucine and photo-methionine allow identification of protein-protein interactions in living cells|journal=Nature Methods|volume=2|pmid=15782218|pages=261–268 |doi=10.1038/nmeth752] In this method, cells are grown with photoreactive diazirineanalogs to leucineand methionine, which are incorporated into proteins. Upon exposure to ultraviolet light, the diazirines are activated and bind to interacting proteins that are within a few angstroms of the photo-reactive amino acid analog.
Tandem affinity purification(TAP) method allows high throughput identification of protein interactions. In contrast to Y2H approach accuracy of the method can be compared to those of small-scale experiments (Collins et al., 2007) and the interactions are detected within the correct cellular environment as by co-immunoprecipitation. However, the TAP tag method requires two successive steps of protein purification and consequently it can not readily detect transient protein-protein interactions. Recent genome-wide TAP experiments were performed by Krogan et al., 2006 and Gavin et al., 2006 providing updated protein interaction data for yeast organism.
*Chemical crosslinking is often used to "fix" protein interactions in place before trying to isolate/identify interacting proteins. Common crosslinkers for this application include the non-cleavable
NHS-estercrosslinker, "bis"- sulfosuccinimidyl suberate(BS3); a cleavable version of BS3, dithiobis(sulfosuccinimidyl propionate)(DTSSP); and the imidoestercrosslinker dimethyl dithiobispropionimidate(DTBP) that is popular for fixing interactions in ChIPassays.
Quantitative immunoprecipitation combined with knock-down(QUICK) relies on co-immunoprecipitation, quantitative mass spectrometry( SILAC) and RNA interference(RNAi). This method detects interactions among endogenous non-tagged proteinscite journal|doi=10.1038/nmeth972| author=Selbach, M., Mann, M. |date=2006 |title=Protein interaction screening by quantitative immunoprecipitation combined with knockdown (QUICK) |journal=Nature Methods |volume=3| pmid=17072306 |pages=981–983] . Thus, it has the same high confidence as co-immunoprecipitation. However, this method also depends on the availability of suitable antibodies.
Dual Polarisation Interferometry(DPI) can be used to measure protein-protein interactions. DPI provides real-time, high-resolution measurements of molecular size, density and mass. While tagging is not necessary, one of the protein species must be immobilized on the surface of a waveguide.
Protein-protein docking, the prediction of protein-protein interaction based on the three-dimensional protein structures only is not satisfactory As of 2006.cite journal|author=Bonvin AM|title=Flexible protein-protein docking| journal=Current Opinion in Structural Biology| date=2006|volume=16| pmid=16488145|pages=194–200|doi=10.1016/j.sbi.2006.02.002] cite journal| author=Gray JJ| title=High-resolution protein-protein docking| journal=Current Opinion in Structural Biology|date=2006| volume=16| pmid=16546374|pages=183–193| doi=10.1016/j.sbi.2006.03.003]
Static Light Scattering(SLS) measures changes in the Rayleigh scatteringof protein complexes in solution and can non-destructively characterize both weak and strong interactions without tagging or immobilization of the protein. The measurement consists of mixing a series of aliquots of different concentrations or compositions with the anylate, measuring the effect of the changes in light scattering as a result of the interaction, and fitting the correlated light scattering changes with concentration to a model. Weak, non-specific interactions are typically characterized via the second virial coefficient. This type of analysis can determine the equilibrium association constant for associated complexes. [cite journal|author=Arun K. Attri and Allen P. Minton| title=Composition gradient static light scattering: A new technique for rapid detection and quantitative characterization of reversible macromolecular hetero-associations in solution|journal=Analytical Biochemistry| volume=346| date=2005| pmid=16188220| pages=132–138| doi=10.1016/j.ab.2005.08.013] .
*Chemical crosslinking followed by high mass
MALDI mass spectrometrycan be used to analyze intact protein interactions in place before trying to isolate/identify interacting proteins. This method detects interactions among non-tagged proteins and is available from CovalX.
*SPINE (Strep-protein interaction experiment) [cite journal| author=Herzberg C., Weidinger LA., Dörrbecker B., Hübner S., Stülke J. and Commichau FM. |date=2007 |title=SPINE: A method for the rapid detection and analysis of protein-protein interactions "in vivo"|journal=Proteomics|volume=7(22)|pmid=17994626|pages=4032–4035 |doi=10.1002/pmic.200700491] uses a combination of reversible crosslinking with formaldehyde and an incorporation of an affinity tag to detect interaction partners "in vivo".
Surface plasmon resonancecan be used to measure protein-protein interaction.
Fluorescence correlation spectroscopy, one protein is labeled with a fluorescent dye and the other is left unlabeled. The two proteins are then mixed and the data outputs the fraction of the labeled protein that is unbound and bound to the other protein, allowing you to get a measure of KD and binding affinity. You can also take time-course measurements to characterize binding kinetics. FCS also tells you the size of the formed complexes so you can measure the stoichiometry of binding.
Protein-protein interaction network visualization
Visualization of protein-protein interaction networks is a popular application of
scientific visualizationtechniques. Although protein interaction diagrams are common in textbooks, diagrams of whole cell protein interaction networks were not as common since the level of complexity made them difficult to generate. One example of a manually produced molecular interaction map is Kurt Kohn's 1999 map of cell cycle control. [cite journal | url=http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pubmed&pubmedid=10436023 | journal=Molecular Biology of the Cell|date=1999|volume=10|title=Molecular Interaction Map of the Mammalian Cell Cycle Control and DNA Repair Systems|author=Kurt W. Kohn| pmid=10436023|pages=2703–2734] Drawing on Kohn's map, in 2000 Schwikowski, Uetz, and Fields published a paper on protein-protein interactions in yeast, linking together 1,548 interacting proteins determined by two-hybrid testing. They used a force-directed (Sugiyama) graph drawing algorithm to automatically generate an image of their network. [cite journal| url=http://igtmv1.fzk.de/www/itg/uetz/publications/Schwikowski2000.pdf | publishersurl=http://www.nature.com/nbt/journal/v18/n12/full/nbt1200_1257.html | volume=18|author=Benno Schwikowski1, Peter Uetz, and Stanley Fields|journal=Nature Biotechnology| title=A network of protein−protein interactions in yeast|date=2000|pmid=11101803| pages=1257–1261| doi=10.1038/82360] [Rigaut G, Shevchenko A, Rutz B, Wilm M, Mann M, Seraphin B (1999) A generic protein purification method for protein complex characterization and proteome exploration. Nat Biotechnol. 17:1030-2. PMID 10504710] [Prieto C, De Las Rivas J (2006). APID: Agile Protein Interaction DataAnalyzer. Nucleic Acids Res. 34:W298-302. PMID 16845013] .
An experimental view of Kurt Kohn's 1999 map [http://www.maplib.net/map.php?id=1700&lat=-52.67138590320257&lng=34.3817138671875&z=9 gmap] . Image was merged via gimp 2.2.17 and then uploaded to maplib.net
Protein-protein interaction prediction
Protein-protein interaction screening
BioGRID, a public repository for protein and genetic interactions
Database of Interacting Proteins(DIP)
NCIBINational Center for Integrative Biomedical Informatics
Protein nuclear magnetic resonance spectroscopy
Protein-protein interaction databases
* [http://hprd.org HPRD Human Protein Reference Database] a (manually) curated database of human protein information with visualization tools.
* [http://www.ebi.ac.uk/intact IntAct Interaction Database] is a public repository for manually curated molecular interaction data from literature.
* [http://dip.doe-mbi.ucla.edu/ DIP]
Database of Interacting Proteins, a manual and automatic catalog of experimentally determined interactions between proteins.
* [http://mimi.ncibi.org MiMI Michigan Molecular Interaction index] integrates data from multiple sources.
* [http://mint.bio.uniroma2.it/mint/ MINT Molecular INTeraction Database] focuses on experimentally verified protein interactions mined from the scientific literature by expert curators.
* [http://mips.gsf.de/proj/ppi/ MIPS Mammalian Protein-Protein Interaction Database]
* [http://www.thebiogrid.org/ BioGRID]
* [http://mippi.ornl.gov/ Microbial Protein-Protein Interactions (MiPPI)]
* [http://bond.unleashedinformatics.com/Action? Biomolecular Interaction Network Database (BIND)]
* [http://www.jcvi.org/mpidb The Mirrobial Protein Interaction Database] integrates microbial interactions and curates microbial interactions from the scientific literature.
Interaction network software
* [http://bioinfow.dep.usal.es/apid/ APID Agile Protein Interaction DataAnalyzer] is an interactive bioinformatics web tool to explore and analyze in a unified and comparative platform main currently known information about protein–protein interactions.
* [http://bioinfow.dep.usal.es/apid/apid2net.html APID2NET: unified interactome graphic analyzer] is an open access tool, included in Cytoscape, that allows to surf unified interactome data by quering APID server and facilitates dynamic analysis of the protein-protein interaction networks.
* [http://www.cytoscape.org Cytoscape] is an open source bioinformatics software platform for visualizing molecular interaction networks and integrating these interactions with gene expression profiles and other state data.
* [http://ophid.utoronto.ca/navigator NAViGaTOR]
* [http://www.molecularconnections.com/products.html#netpro NetPro] Is a comprehensive fully hand-curated knowledgebase of Protein-Protein, Protein-Small molecules DNA and RNA interactions.
* [http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pubmed&pubmedid=12620107 Osprey]
* [http://www.celeader.com/genfiles/CELeadercomPA8_05124356.asp?id=1.2&ln=l12 PA 800 Protein Characterization System] uses Capillary Electrophoreses technology to determine a protein's molecular weight, resolve differences in iso electric point, generate high-resolution peptide maps and carbohydrate profiles, and provide front-end separation and introduction of these proteins to mass spectrometry.
* [http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=368431 VisANT]
* [http://www.yworks.com/en/products_yed_about.htm yEd] , graph editor.
* [http://www.graphviz.org/ Graphviz]
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