Experimentally Identifying Protein Interactions Biology Essay

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Mammalian cells encapsulate a variety of critical processes ranging from DNA-replication to signal transduction. The vitality of the occurrence of successful processes lies in the interactions of proteins. Proteins being made of amino acids are quite varied, and the amino acid composition an individual protein sets it and its interaction different to others. The associations present between two protein molecules are one of the essential constituent of the processes alongside protein-ligand and other molecular interactions. Protein can bind to other proteins in a variety of ways. The interactions can be stable or ephemeral depending on where they are localised and the function they aim to execute. In order to understand protein recognition and its cellular interaction a large number of experimental methods have been developed. The methods vary, as some simply distinguish the presence of proteins, some screen for interactions and others characterize the protein structurally and functionally.

These protein-protein interaction exhibit an assortment of characteristics enabling successful observation via measurement. They range from altering the kinetic proteins of the proteins involved, allowing substrate channelling, forming a new binding site, changing the specificity of the protein and so in the end inactivating the protein.

The choice of experimental procedures depends on whether the identity of the proteins present is known and on the features of the proteins or the interactions being looked at, whether structural or functional. Firstly in order to test for the presence of a protein- protein interaction, an initial screening will have to be done. For this, experimental procedures such as a Label transfer or a Two-Hybrid screen (Yeast two-hybrid) can be carried out, which would illustrate the presence of even fragile, short-lived interactions.

Label transfer works on the basis of a 'bait and prey' theory, in which a complex consisting of a tag such as biotin and a concealed cross linking agent is used (4). This complex is in turn able to bind to the protein via cleavage of a linker present on the protein. Only after the protein complex is able to integrate itself into is stable conformation, the section responsible for the cross-linking starts up (the denaturing step) removing the linker moiety and leaving the protein tagged, as shown in fig1.

Fig. Mechanism of Label transfer

Adapted from Bo Liu, Chase T. Archer, Lyle Burdine, Thomas G. Gillette, and Thomas Kodadek (2007) Label Transfer Chemistry for the Characterization of Protein-Protein Interactions. J Am Chem Soc.,Pubmed http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2529226/

The yeast two- hybrid is a variation of the two-hybrid system (4), that uses genetic-based organisms, for example yeast as a host, hence allowing detection of a list of interacting proteins and a number of unknown proteins. Its activity is based on a paradigm similar to the 'bait and prey', but utilizes the modular attribute of the transcription factors present in eukaryotes. An exemplar factor that is used, GAL4, consists of two independent domains DNA-binding domain (DNA-BD) and a transactivating domain (AD). This modular nature of the transcription factors is common amongst eukaryotes (2). Testing for an interaction between two proteins can be conducting by binding of the proteins to either of the domains. Protein 1 can be bound to the DNA binding creating the 'Bait' and protein 2 to the AD creating the 'Prey', before which enzymatic slicing occurs at the N-terminal of the DNA-BD and the C-terminal of the AD to remove the sequence in-between (depicted in part A, Fig 2) . If an intracellular association between the fusion proteins occurs, the bait and prey are co-expressed(1), as both the domains are brought together leading to a successful functioning Gal4 gene that initiates transcription by binding to a promoter of a reporter gene (part c, Fig 2 )

Fig 2. Yeast-2-hybrid action mechanism for protein-protein interaction analysis

Adapted from Friedrich Marks, Ursula Klingmuller, Karin Muller-Decker. Cellular Signal Processing pg. 191 figure 5.8

Hence it allows the protein-protein interaction to be identified by measurement of the reporter gene activity.

Affinity Chromatography is an important method for protein-protein interaction determination. It uses the basic concept of the affinity of one molecule to another to elute one binding partner with the help of an immobilised binding partner in a complex solution mixture. The immobilised protein becomes a marker to find the binding protein in the heterogeneous mixture (1). This procedure is an entrapment of the protein via a reversible interaction between a protein or a group of proteins and a specific ligand protein joined to the column matrix. The interaction present between proteins can be weak or strong depending on the type of interactions involved, which can range from hydrophobic to van der Waals and hydrogen bonding. This interaction has to be reversed in order to elute the solute of interest.

Fig 3. Mechanism for Affinity Chromatography of proteins.Adapted From: http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/A/AffinityChrom.gifFirstly, an immunoadsorbant containing a solid matrix, such as agrose, or sephadex, which has the ligand adhered covalently to it, is made. Secondly, the mixture containing the proteins is poured in and passed over the matrix, during which the binding partner proteins to the ligands, will bind non-covalently creating an inert matrix. Thirdly, all other proteins without specificity for the ligand protein used will pass through and be eluted first (1). The secondary elutions would contain proteins that are weakly attached to the ligand protein. This mechanism is depicted in fig 3. The strongly retained protein would be retained via use of cofactors or altered high salt concentrations. For successful protein elution to be obtained a purified protein should be initially used which would prevent impurities attaching (2). The dissociation coefficient of the protein-protein interactions is crucial for a successful detection; specifically the ligand protein concentration has to be above the coefficient to detect the interaction. Hence in the case of a weak protein-protein interaction the concentration of the protein acting as the ligand has to be kept high. Another feature that aids detection is protein labelling prior to chromatography. Finally the binding partner proteins can be eluted and detected via the use of western blotting. Through the use of affinity chromatography the protein interaction's strength can be obtained (4).

Methods that personify the proteins functioning in the protein-protein interactions are also widely present. Surface Plasmon Resonance spectroscopy is a means of kinetic and dynamic characterisation of complex physical interactions between proteins based on the excitatory effect of surface plasmons by light (2). Simplified this method observes the adsorption of the substance in question onto metal surfaces like gold or silver, that are known to be planar. It focuses on complex formation via observing the resonance angle of light on a planar surface caused due to alteration in the surface's refractive index,(fig 4) which gets noted by diode array detector. Hence, this procedure can be used to measure interactions between two or more biological molecules such as proteins, due to their refractive indexes being same.

Following the principle, one of the proteins is immobilised on to a dextran polymer (fig 4), the sensor chip surface, and acts as the ligand. The second protein on the other hand is flowed through a dextran walled cell, in a buffer solution. In the case of an interaction occurring, the secondary binding partner protein is retained on the sensor surface, the build up of which causes a change in the resonance angle of light in turn altering the refractive index.

The effects of the protein-protein interactions can be readily measured because of the increasing protein concentration near the surface affecting the resonance angle. Following this the refractive index is measured over time by a sensorgram, which in turn illustrate the strength of the interaction and its kinetics, hence hinting on possible biological function.

The strength of the interaction could illustrate possible type environment, whilst the kinetics could illustrate the type of reaction.

This method supports the use of a solution containing an unknown protein, as the immobilised protein can be used as marker for a binding partner present in the mixture of protein flowing through the cell.

Fig4. Method of Surface Plasmon Resonance spectroscopy

Adapted from; Institute of Structural Biology and Biophysics (ISB)

Cross linking is a flexible method that aids protein protein interaction detection and also allows determination of structural design. Sequentially it also allows distinguishing proteins that specifically interacts to a given protein. Chemical cross-linking primarily repairs protein-protein interactions. By using two known individual proteins along side a chemical cross linker, it becomes evident on the SDS gel if an interaction has been induced between the two proteins.

Distinguishing protein protein interactions is based on the complexity of the cross linking patterns obtained. Initiation of this procedure takes place following the cleaving of the protein by a bifunctional reagent RSSR. Then SDS gel based fractionation of the protein occurs, separating the protein based on the molecular weights. The RSSR bound proteins, the proteins between which an interaction is present fractionate at the heavier molecular weighted regions. A secondary SDS gel is then conducted in the presence of a reducing agent, which cleaves the S-S bond, hence causing the proteins with interactions to migrate off the diagonal. Hence the molecular weights and the size of the cross linkage can be obtained from this method can illustrate the structural design of the proteins. This enables analysis of the subunit structure of the interactions present and its relation to the function.

In order to identify the presence of a protein- protein interaction a method involving labelling is quite useful. The use of a labelled protein as a probe allows prominent detections with both transient and stable interactions. The probe is used to screen an expression library of cDNA, during which interactions would occur on the nitrocellulose membrane between the probe and a protein, which has been put through the immobilisation process. The identity of the probe can be varied (transcription factors) to check for interactions which would illustrate the presence of the protein amidst the biological process, thus deciphering its biological function. The presence of domains, such as the leucine zipper which is DNA-binding or the zinc binding domain, could also be used in case of which a successful interaction would mean the domains are present in the proteins, and their functions could then be looked up.

Finally, Fluorescence Correlation Spectroscopy provides ample support for protein-protein interaction detection via fluorescence tagging. It is a procedure that examines the variation in the fluorescence in a system and relates them to events in the molecule. FCS quantifies the interactions between proteins based on the molecular weights of the components, which separates the free proteins from the ones in complex with other proteins.(3) It focuses a laser into the diffraction limited spot of a sample abundant in particles that fluoresce. The fluorescence obtained is a measure of the number of particles that fluoresce in the detection volume. This method in turn provides the direct concentrations of the proteins present along with the distinction of bound and unbound. FSC also provides information on the binding kinetics of the proteins and their affinities for binding. Observing the change in concentrations, the procedure can obtain information regarding the stoichiometry of each of the protein in the complex.(3) On the whole FSC is a very good method for protein-protein interaction detection method as it does not require protein immobilisation, hence obtaining information on the interaction whilst the proteins are freely present. Therefore, it provides information that relates better to biological functions of the interactions.

Through the use of a mix of these methods, the interaction sites for the proteins could be mapped. A detailed map of the biomolecular interaction networks the proteins could be generated which would aid identification of unknown protein information, based on similarities with other proteins. These are only some of the methods from the variety present for protein-protein interaction identification and characterisation.

Therefore, the experimental methods used can be based on whether the proteins depicting the interactions are known or unknown. If the proteins involved in the interactions are unknown initial screening procedures can be implemented, such as affinity purification that would depict the strength and the ligand specificity for the interaction. It would also illustrate the complexity of the proteins present. Following this other methods such as protein probing could be implemented, which illustrates the cellular presence, hence depicting the biological function of the proteins involved. Methods such as Surface Plasmon Resonance spectroscopy and FCS would specifically show structural, biological, kinetic and dynamic significance of the complex. On the whole if used together the variety of methods would yield the structure, function, localisation and features relating to its biological significance.