Biology The Thermodynamics of Protein

Biology: The Thermodynamics of Protein-Protein Interactions Essay There are various methods that have been developed over these years to study protein-protein interactions (PPIs). PPI plays a big role in the cell-signalling cascade; for instance, dephosphorylation of glycogen synthase by protein phosphatase-1 results in glycogen synthesis. To know whether a specific protein binds to its partner, for example, whether TFIIH interacts with TFIIE or TFIIF to complete the pre-initiation complex in transcription, different methods such as co-immunoprecipitation (co-IP), glutathione-S-transferase (GST) pull down assays, yeast-two-hybrid (Y2H) assays, isothermal titration calorimetry (ITC), surface plasmon resonance (SPR), nuclear magnetic resonance (NMR) spectroscopy and etc. can be use to validate PPIs. Yet, doing one experiment using one method is not enough to validate the PPI between two or more proteins. Factors such as overexpression of proteins and manipulation of the agents used in the experiment could result in a bias data. Thus, the results shou ld be unbiased by incorporating different methods in the experiment to validate the PPI. In this essay, the different methods will be described and the factors that cause the different methods giving rise to different results will be discussed. Co-IP is the most commonly used methods to verify protein-protein interactions (Berggà ¥rd et al., 2007). Antibodies that are specific to the bait complexes are used to capture the bait complexes in a cell lysate shown in Fig. 1. The antibody is immobilized on Protein A/G, which is covalently bound to the agarose beads. Since the antibody is specific to only the bait complex, the antibody will not bind to other proteins found in the cell lysate, and hence, these proteins will be wash off. The antibody-bait complex can be eluted after washing. The bound proteins in the bait complex can be identified by using mass spectrometry (MS) or by immunoblotting (Berggà ¥rd et al., 2007). One major disadvantage of co-IP is the tendency of washing off interacting proteins together with unbound proteins, affecting the experiment. One recent study have shown to overcome this by introducing two-step chemical crosslinking by co-IP coupled with tandem MS to identify PPIs, and also to allow better st udy on weakly bound PPI (Huang Kim, 2013). GST pull-down assay is an in vitro method that is widely used to purify specific protein in a cell lysate, and the recombinant protein is often overexpressed in the cell to aid in the purification. GST fusion proteins are commonly expressed from E. coli and being purified through immobilized glutathione-coated beads matrix (Panchenko Przytycka, 2008). Only proteins that contains GST-tagged will be able to bind to the matrix and unbound proteins will be washed off. Once GST fusion protein bound to the matrix, the prey protein solution can be added to matrix and only those proteins that interact with GST fusion protein will bind to the GST fusion protein on the matrix and unbound proteins will be washed off as shown in Fig. 2 (Panchenko Przytycka, 2008). The yeast-two-hybrid (Y2H) system is based on the idea that transcription factor have two distinct functional domains that can be spliced into two, the DNA binding domain (BD) that binds to the upstream activating sequences (UAS) and an activating domain (AD) which activates transcription (Osman, 2004). Without the presence of either domain, transcription of the gene cannot take place. However, if both domains are placed close to each other, it is enough to restore a functional transcription factor and thus, activating transcription of a reporter gene (Osman, 2004). In Y2H system, there are two plasmids being constructed. The first plasmid contains the bait protein genomic sequence fused to BD sequence and the second plasmid contains the prey protein sequence being fused to the AD sequence (Berggà ¥rd et al., 2007). Both plasmids are inserted to the yeast cell, where the bait-BD protein and prey-AD protein are targeted to the nucleus. The Y2H system is described in the legend of Fig . 3. One major advantage of using Y2H system is the ability to detect PPIs in vivo as compared to co-IP and GST-tags. However, Y2H system often results in the data having large number of false positives, causing the protein interactions that are being identified to be unreliable or is questionable (Deane et al., 2002). Hence, it is important to validate the protein interactions identified in Y2H using other validation methods. Essay on How does Heat Transfer WorksWorks Cited Berggà ¥rd T., Linse S. and James P. (2007). Methods for the detection and analysis of protein-protein interactions. 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