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Information about Protein Protein Interaction

Protein-protein interactions refer to the association of protein molecules and the study of these associations from the perspective of biochemistry, signal transduction and 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 signalling 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 cytoplasm to nucleus or vice versa in the case of the nuclear pore importins), or a protein may interact briefly with another protein just to modify it (for example, a protein kinase will 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 acid tyrosine. 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 sensitivity and specificity of 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-immunoprecipitation is 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.
  • 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. A pull-down assay is distinct from immunoprecipitation in that it uses some ligand other than an antibody to capture the protein complex.
  • Label transfer can 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.
  • The yeast two-hybrid screen 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 [1] In this method, cells are grown with photoreactive diazirine analogs to leucine and 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) detects interactions within the correct cellular environment (e.g. in the cytosol of a mammalian cell) (Rigaut et al., 1999). This is a big advantage compared to the yeast two-hybrid approach. However, the TAP tag method requires two successive steps of protein purification. Thus, it can not readily detect transient protein-protein interactions.
  • 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-ester crosslinker, bis-sulfosuccinimidyl suberate (BS3); a cleavable version of BS3, dithiobis(sulfosuccinimidyl propionate) (DTSSP); and the imidoester crosslinker dimethyl dithiobispropionimidate (DTBP) that is popular for fixing interactions in ChIP assays.
  • 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 proteins[2]. 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.
  • As of 2006 available methods of Protein-protein docking, the prediction of protein-protein interaction based on the three-dimensional protein structures only, are not satisfactory.[3][4]
  • Static Light Scattering (SLS) is a sensitive and precise measure of weight-averaged molar mass that can non-destructively characterize both weak and strong interactions without tagging or immobilization of the protein. The measurement consists of introducing a series of aliquots of different concentrations and/or compositions into the detector, and fitting the resultant signals to equations representing the effect of the interaction on the light scattering. Weak, non-specific interactions are typically characterized via the second virial coefficient. Stronger interactions with specific stoichiometries, manifesting as reversible homo- and hetero-associations, are analyzed by fitting the signals to association models. The analysis determines the equilibrium association constant for each associated complex present in the solution[5]. Association kinetics may also be characterized via SLS. A system incorporating a static light scattering detector, an on-line concentration detector, and automation of the sample preparation, delivery, and data acquisition, as well as analysis of virial coefficients and reversible protein associations, is available from Wyatt Technology Corp..
  • Chemical crosslinking followed by high mass MALDI mass spectrometry can 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.

Protein-protein interaction network visualization

Visualization of protein-protein interaction networks is a popular application of scientific visualization techniques. 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.[6] 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.[7]

An experimental view of Kurt Kohn's 1999 map gmap. Image was merged via gimp 2.2.17 and then uploaded to maplib.net

See also

References

  • 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.
  • Selbach M, Mann M. (2006) Protein interaction screening by quantitative immunoprecipitation combined with knockdown (QUICK). Nat Methods. 3:981-3. PMID: 17072306.
  • Prieto C, De Las Rivas J (2006). APID: Agile Protein Interaction DataAnalyzer. Nucleic Acids Res. 34:W298-302. PMID: 16845013.

References

1. ^ Suchanek, M., Radzikowska, A., and Thiele, C. (2005) Photo-leucine and photo-methionine allow identification of protein-protein interactions in living cells. Nature Methods. 2, 261 – 268.
2. ^ Selbach, M., Mann, M. (2006) Protein interaction screening by quantitative immunoprecipitation combined with knockdown (QUICK). Nature Methods. 3, 981 - 983.
3. ^ Bonvin AM. Flexible protein-protein docking. Curr Opin Struct Biol. 2006; 16: 194-200.
4. ^ Gray JJ. High-resolution protein-protein docking. Curr Opin Struct Biol. 2006; 16: 183-193.
5. ^ K. Kameyama and A.P. Minton Rapid quantitative characterization of protein interactions by composition gradient static light scattering. Biopys. J. 2006; 90: 2164-2169
6. ^ [1] Mol Biol Cell. 1999
7. ^ [2] Nature 2000

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Proteins are large organic compounds made of amino acids arranged in a linear chain and joined together by peptide bonds between the carboxyl and amino groups of adjacent amino acid residues.
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Biochemistry is the study of the chemical processes in living organisms.[1] The word "biochemistry" comes from the Greek word βιοχημεία biochēmeia, which means "the chemistry of life.
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In biology, signal transduction refers to any process by which a cell converts one kind of signal or stimulus into another, most often involving ordered sequences of biochemical reactions inside the cell, that are carried out by enzymes, activated by second messengers resulting in
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graph theory is the study of graphs; mathematical structures used to model pairwise relations between objects from a certain collection. A "graph" in this context refers to a collection of vertices or 'nodes' and a collection of edges
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In biology, signal transduction refers to any process by which a cell converts one kind of signal or stimulus into another, most often involving ordered sequences of biochemical reactions inside the cell, that are carried out by enzymes, activated by second messengers resulting in
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protein complex is a group of two or more associated proteins formed by protein-protein interaction that is stable over time. Protein complexes are a form of quaternary structure.
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Cytoplasm is a gelatinous, semi-transparent fluid that fills most cells. Eukaryotic cells contain a nucleus that is kept separate from the cytoplasm by a double membrane layer. The cytoplasm has three major elements; the cytosol, organelles and inclusions.
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nucleus (3) ribosome (4) vesicle (5) rough endoplasmic reticulum (ER) (6) Golgi apparatus (7) Cytoskeleton (8) smooth ER (9) mitochondria (10) vacuole (11) cytoplasm (12) lysosome (13) centrioles]]

In cell biology, the nucleus (pl.
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Nuclear pores are large protein complexes that cross the nuclear envelope, which is the double membrane surrounding the eukaryotic cell nucleus. There are about on average 2000 nuclear pore complexes in the nuclear envelope of a vertebrate cell, but it varies depending on the
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protein kinase is a kinase enzyme that modifies other proteins by chemically adding phosphate groups to them (phosphorylation). This class of protein is further separated into subsets such as PKC alpha, PKC beta, and PKC gamma, each with specific functions.
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Src homology 2 domain (or SH2 domain) is a protein domain of about 100 amino acid residues first identified as a conserved sequence region among the oncoproteins Src and Fps.
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amino acid is a molecule that contains both amine and carboxyl functional groups. In biochemistry, this term refers to alpha-amino acids with the general formula H2NCHRCOOH, where R is an organic substituent.
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Tyrosine (abbreviated as Tyr or Y)[1] or 4-hydroxyphenylalanine, is one of the 20 amino acids that are used by cells to synthesize proteins. It is a non-essential amino acid and it is found in large quantities in casein.
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Sensitivity may refer to:
  • Sensitivity (biology)
  • Sensitivity (electronics)
  • Sensitivity (human)
  • Sensitivity (information)
  • Sensitivity (tests, medicine)
  • Sensitivity (radio receiver)

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Specificity may refer to:
  • Specificity (tests), a measure of a test's effectiveness
  • A concept relating to Cascading Style Sheets

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Immunoprecipitation (IP) is the technique of precipitating an antigen out of solution using an antibody specific to that antigen. This process can be used to enrich a given protein to some degree of purity.
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Antibodies (also known as immunoglobulins) are proteins that are found in blood or other bodily fluids of vertebrates, and are used by the immune system to identify and neutralize foreign objects, such as bacteria and viruses.
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A western blot (alternately, immunoblot) is a method to detect a specific protein in a given sample of tissue homogenate or extract. It uses gel electrophoresis to separate native or denatured proteins by the length of the polypeptide (denaturing conditions) (Figure
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Fluorescence resonance energy transfer (FRET) describes an energy transfer mechanism between two chromophores.

A donor chromophore in its excited state can transfer energy by a nonradiative, long-range dipole-dipole coupling mechanism to an acceptor chromophore in close
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In chemistry, a ligand is an atom, ion, or molecule (see also: functional group) that generally donates one or more of its electrons through a coordinate covalent bond to, or shares its electrons through a covalent bond with, one or more central atoms or ions (these ligands act as
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A. Gal4 transcription factor gene produces two domain protein (BD and AD) which is essential for transcription of the reporter gene (LacZ).
B,C. Two fusion proteins are prepared: Gal4BD+Bait and Gal4AD+Prey.
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Immunoprecipitation (IP) is the technique of precipitating an antigen out of solution using an antibody specific to that antigen. This process can be used to enrich a given protein to some degree of purity.
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Leucine (abbreviated as Leu or L)[1] is an α-amino acid with the chemical formula HO2CCH(NH2)CH2CH(CH3)2. It is an essential amino acid, which means that humans cannot synthesise it.
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Methionine (abbreviated as Met or M)[1] is an α-amino acid with the chemical formula HO2CCH(NH2)CH2CH2SCH3. This essential amino acid is classified as nonpolar.
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1 ångström =
SI units
010−12 m 010−3 nm
Natural units
01024 lP 0 a0
US customary / Imperial units
010−12 ft 010−9
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The Tandem Affinity Purification (TAP) method involves the fusion of the TAP tag to the C-terminus of the new protein. The TAP tag consists of calmodulin binding peptide (CBP) from the N-terminal, followed by tobacco etch virus protease (TEV protease) cleavage site and Protein A,
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The cytosol (cf. cytoplasm, which also includes the organelles) is the internal fluid of the cell, and a portion of cell metabolism occurs here. Proteins within the cytosol play an important role in signal transduction pathways and glycolysis.
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Mammalia
Linnaeus, 1758

Subclasses & Infraclasses
  • Subclass †Allotheria*
  • Subclass Prototheria
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