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 should 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 (Berggård 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 compl... ... middle of paper ... ...nflammatory Arthritis in Mice. Science. 332 (6028), pp. 478-484. Wissmueller S., Font J., Liew C.W., Cram E., Schroeder T., Turner J., Crossley M., Mackay J.P. and Matthews J.M. (2011). Protein-protein interactions: analysis of a false positive GST pulldown result. Proteins. 79 (8), pp. 2365-2371. Yu H. (1999). Extending the size limit of protein nuclear magnetic resonance. Proceedings of the National Academy of Sciences. 96 (2), pp. 332-334. Zhang X., Tang H., Ye C. and Liu M. (2006). Structure-based drug design: NMR-based approach for ligand-protein interactions. Drug Discovery Today: Technologies. 3 (3), pp. 241-245. Zhou YL., Liao JM., Du F. and Liang Yi. (2005). Thermodynamics of the interaction of xanthine oxidase with superoxide dismutase studied by isothermal titration calorimetry and fluorescence spectroscopy. Thermochimica Acta. 426 (1-2), pp. 173-178.
The results of this experiment showed a specific pattern. As the temperature increased, the absorbance recorded by the spectrophotometer increased indicating that the activity of peroxidase enzyme has increased.At 4C the absorbance was low indicating a low peroxidase activity or reaction rate. At 23C the absorbance increased indicating an increase in peroxidase activity. At 32C the absorbance reached its maximum indicating that peroxidase activity reached its highest value and so 32 C could be considered as the optimum temperature of peroxidase enzyme. Yet as the temperature increased up to 60C, the absorbance decreased greatly indicating that peroxidase activity has decreased. This happened because at low temperature such as 4 C the kinetic energy of both enzyme and substrate molecules was low so they moved very slowly, collided less frequently and formed less enzyme-substrate complexes and so little or no products. Yet, at 23 C, as the temperature increased, enzyme and substrate molecules
n.d. - n.d. Peptides and Proteins. Proteins. Retrieved July 25, 2008, from http://www.cd http://www.cem.msu.edu/reusch/VirtualText/protein2.htm Ophardt, C. E. (2003).
This experiment was conducted to determine the effects of pH and temperature on peroxidase from a potato. The optimum temperature for peroxidase was determined to be 23°C, because it had a rate of absorbance of 0.3493, higher than the other temperatures evaluated. A temperature of 48°C is inefficient of speeding up peroxidase activity because its rate of absorbance was 0.001.
Introduction: Within this experiment we wish to facilitate a greater understanding of the concepts of experimental design and quantifying techniques. Specifically, this lab will allow us to gain an enhanced understanding of the isolation of a protein using differential solubility, which allows us to separate and purify various proteins using high concentrations of a specific salt so that they may be studied in great detail. Last week we separated our desired protein using ammonium sulfate. Since we have already extracted the desired protein, we will begin quantifying the amount using the Bradford Protein Assay. Because it is a dye-binding assay, we will use the spectrophotometer to measure the absorbance of various dilutions of a protein: this will comprise our standard curve. We will then compare the absorbance of our extracted protein from l...
Protein have connection with amino acid to help in functions of: skin, muscle, hair and bones
How the Concentration of the Substrate Affects the Reaction in the Catalase Inside Potato Cells Introduction Enzymes are made of proteins and they speed up reactions, this means that they act as catalysts. Hydrogen peroxide is a byproduct of our cell's activities and is very toxic. The enzymes in our bodies break down the hydrogen peroxide at certain temperatures they work best at body temperature, which is approximately 37 degrees. At high temperatures, the cells begin to denature. This means that the hydrogen peroxide is prevented from being broken down because they will not 'fit' into the enzyme.[IMAGE] Objective I am going to find out how the concentration of the substrate, hydrogen peroxide affects the reaction in the catalase inside the potato cells.
CP consists of a single domain with high α-helical content [4]. The N-terminal part this domain is surface exposed whereas the C-terminal region buried in the virion. Several experiments indicate the CP is an O-glycoprotein. Equal amounts of galactose and fructose residues are O-linked to an acetylated serine residue at the N-terminal region [2]. This mediates the formation of a structured...
Phosphorylation and dephosphorylation can activate or deactivate a protein but changing in 3-D conformation and as a result changing the ability to interact with other proteins. Just like in Arabidopsis and other an...
For example, some of the proteins contain pleckstrin homology domains that bind phosphoinositide and others contain C2 domains that bind membrane lipids in the presence of Ca2+, some proteins contain positively charged regions that bind to negatively charged phosphoglycerides and others contain covalently attached fatty acyl groups or prenyl groups that anchor them to membranes. Another example is Annexin shows Ca2+ dependent binding to the cytosolic surfaces of cell membranes. Ca2+ ions bind to the iface of each annexin and this promote protein–lipid interactions through a combination of electrostatic and hydrophobic mechanisms. The same result has been shown by crystallographic studies with phosphoglyceride analogs, suggested that some of the bound Ca2+ ions may bind directly to the oxygens of phospholipid head groups. Addition to this, adjacent membrane lipids that do not bind proteins directly may modulate the protein–lipid interactions, the binding of proteins to membrane surfaces may promote further changes in the structure and function of the proteins, and groups of proteins that bind to the same membrane surface may interact with each other to produce complex membrane
“This knowledge will help us design drugs that mimic the viral effects on these proteins to either activate a host’s immune response or shut it down,” said Dr. Michael Gale, associate ...
M proteins: M proteins are found on the surface of the organism and protect it against phagocytosis. The M proteins prevent the attachment of complement proteins to the cell. Complement proteins which are attached to the bacterium “tag” it for destruction by phagocytic cells, such as neutrophils and macrophages, in a process called opsonisation. By inhibiting this process, the M protein allows the group A streptococcus to survive longer...
Figure 3 shows the structure of the prepared protein along with the ligand depicted in green colour.
In the hierarchial organisation of proteins, domains are found at the highest level of tertiary structure. Since the term was first used by Wetlaufer (1973) a number of definitions exist reflecting author bias, however all of the definitions agree that domains are independently folding compact units. Domains are frequently coded by exons and therefore have specific functionality. Among the many descriptions of protein domains the two most striking and simple are " Protein evolutionary units" and "Basic currency of Proteins".
There are four main levels of a protein, which make up its native conformation. The first level, primary structure, is just the basic order of all the amino acids. The amino acids are held together by strong peptide bonds. The next level of protein organization is the secondary structure. This is where the primary structure is repeated folded so that it takes up less space. There are two types of folding, the first of which is beta-pleated sheets, where the primary structure would resemble continuous spikes forming a horizontal strip. The seco...
Proteins are considered to be the most versatile macromolecules in a living system. This is because they serve crucial functions in all biological processes. Proteins are linear polymers, and they are made up of monomer units that are called amino acids. The sequence of the amino acids linked together is referred to as the primary structure. A protein will spontaneously fold up into a 3D shape caused by the hydrogen bonding of amino acids near each other. This 3D structure is determined by the sequence of the amino acids. The 3D structure is referred to as the secondary structure. There is also a tertiary structure, which is formed by the long-range interactions of the amino acids. Protein function is directly dependent on this 3D structure.