A Study of Nonentein


Protein-protein interactions are extremely important to the biological functions of all proteins. When a new protein is discovered, finding out which proteins will interact with it, and which of these occur in the cell can reveal a huge amount of information about the discovered protein. This could be some of the proteins roles or functions, or simply just more information about the surface structure of the protein. This essay is split into two main sections: firstly, some of the techniques used to discover which proteins would interact with the fictitious nonentein, and secondly, methods used to find out which of these occur normally inside a cell and are required for nonentein's function. Some techniques such as co-immunoprecipitation can be used for both.

Protein-Protein Interactions

One of the most widely praised technique for discovering which proteins interact with each other is known as the yeast two hybrid system. Proteins have domains which allow them to bind to certain things in order to perform a certain function. In the yeast two hybrid system, the first protein, in this case nonentein binds to the promoter section of an RNA strand which codes for the expression of a gene, and is known as the ‘bait' (K.H. Young, 1998). Then, if the tested protein interacts with the nonentein then it will bind to it and is known as the ‘prey'. Both proteins have now combined to form what is called a transcriptional activator (TA) (Sobhanifar, S, 2003) which then binds with polymerase, moves along the strand and the gene is expressed as a result. The expression of the gene will tell us that the second protein is a protein that interacts with nonentein. If it is not a protein which has interactions with nonentein then the gene will not be expressed.

Another very commonly used technique for discovering proteins which interact with each other is co-immunoprecipitation. In this method, an antibody with one end attached to a larger molecule (such as a protein A-agarose bead shown in figure 2) is used which attaches to the known protein. Any proteins which interact with the known protein will bind to the known protein's domain (surface) and will therefore also be joined to the antibody. (ed: Haian Fu, 2004) Western blotting, which is a gel electrophoresis technique, can then be done to separate the antibody-protein complex from the other proteins in the cell, and the unknown protein can then be identified. One small flaw in the co-immunoprecipitation technique is that if two unknown proteins become attached to the antibody complex, then it does not specify whether both of the unknowns are interacting directly to the known protein, or if the second protein is also interacting with a unrelated third protein.

There is another technique that is in many ways similar to co-immunoprecipitation. It is called Tandem Affinity Purification (TAP) and involves the fusing of a TAP tag to usually the C terminus of the known protein. This complex can be purified out using an Immunoglobulin G matrix and washing, pulling with it any attached proteins ( Puig, O, et al 2002). The attached proteins can then be identified. However, it is possible that the TAP tag added to the protein might prevent some naturally interacting proteins to bind to the studied protein.

Which Interactions occur in a Cell

The Database of Interacting Proteins(DIP) is a good reference to show the complexity of discovering which interactions occur normally in a cell. Figure 3 shows how the database can be used to identify the likely interactions that occur in a cell. (Xenarios I, et al, 2002)

How complex protein protein interactions can become and how many possibilities there are. The yellow circles are other proteins that can interact with the central protein (calmodulin in this case) and the thickness of the lines towards the central protein indicate the confidence that the two proteins naturally interact. The thicker the line, the more chance there is that the proteins interact inside a cell.

However, because nonentein is newly discovered, there will be no collected information on its interactions to utilise. Therefore, experiments must be done to identify the proteins that show greatest relation with nonentein as these will be the proteins that interact with nonentein naturally, (i.e the proteins that would have the thickest lines on figure 3 if the central protein was nonentein). One such experiment uses the genome sequence to find proteins that interact with each other. This can be done because some interacting proteins have very similar genetic codes, some that only differ in the number of repeating units, and in some organisms can fuse to a single protein (Marcotte, M, et al, 1999). This method is not sufficient to enable the creation of a diagram such as figure 3, other methods are needed to obtain enough information about the protein interactions to be confident to say which occur in the cell and are required for nonentein's function.

Co-immunoprecipitation can be one of the other methods as the procedure occurs inside the cell where the known protein is found, and therefore the interacting proteins that are collected, would interact naturally. However, the flaw with this method stated above in the previous section might still cause problems. If enough information is gathered on nonentein's interactions with other proteins, especially those that occur inside the cell, then it is possible to predict the interactions that aid nonentein in carrying out its function. A protein-protein interaction network (PPI network) can be created. A PPI network is a collection of data, much like figure 3, of a protein's interactions. An identification method known as PPISpan can then be used which looks for patterns in the interactions and recurring protein-protein interactions (Mehmet, T, Tolga, C, 2008). The proteins highlighted by this technique are the most likely to be involved in assisting nonentein's function.


The techniques and methods stated above are just some of a vast amount of ways in which scientists can learn about the interactions of proteins. Some target other proteins that interact directly, others focus on the understanding of the structure and some study the genome sequence for clues to similar proteins. Some of the main methods used such as co-immunoprecipitation and Tandem Affinity Purification have flaws and might not give a completely accurate impression of protein interactions. However, when several techniques are used, a more reliable idea of a proteins interactions can be created, which leads on to the mapping of these interactions and subsequently the predictions through methods such as PPISpan, of the most likely proteins to be involved in the functional process of the studied protein. There is a huge amount that modern day science can learn from the way in which proteins interact and these methods are allowing protein understanding to develop.


Xenarios I, Salwinski L, Duan XJ, Higney P, Kim S, Eisenberg D (2002) DIP: The Database of Interacting Proteins. A research tool for studying cellular networks of protein interactions.

Adams, J,(2008) The Complexity of Gene Expression, Protein Interaction, and Cell Differentiation

Marcotte, E, Matteo Pellegrini, Ho-Leung Ng, Danny W. Rice, Todd O. Yeates, David Eisenberg (1999) Detecting Protein Function and Protein-Protein Interactions from Genome Sequences

Sobhanifar, S, (2003) The Yeast Two-Hybrid Assay: An Exercise In Experimental Eloquence

K.H. Young (1998) Yeast Two-Hybrid: So Many Interactions, (in) So Little Time ...

editor: Haian Fu (2004) Protein-Protein Interactions: Methods and Applications

Mehmet, T, Tolga, C, (2008) Discovering functional interaction patterns in protein-protein interaction networks

Puig, O, Caspary, F, Rigaut, G, Rutz, B, Bouveret, E, Bragado-Nilsson, E, Wilm, M, and Séraphin, B (2002) The Tandem Affinity Purification (TAP) Method: A General Procedure of Protein Complex Purification

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