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.
Abstract/Summary: “Proteins account for more than 50% of the dry weight of most cells, and they are instrumental in almost everything organisms do” (Campbell, 1999). The significance of proteins to the continuation of our biological systems is undeniable, and a study of how to quantify proteins seems an appropriate introduction to our studies of biology. In order to study proteins we must first know how to separate then quantify the amount using basic principles of experimental design such as a standard curve. In this experiment we wish to quantify the amount of previously extracted protein by measuring the absorbance of the unknown amount and determining its concentration by overlaying it against a standard curve of the absorbance of known concentrations of the protein. We used the dye agent Bradford Protein Assay to get an absorbance of 0.078, 0.143, 0.393, 0.473, and 0.527 at the protein’s respective concentrations of 0.28, 0.56, 0.84, 1.12, and 1.40 mg/mL. When a best-fit line was applied to the standard curve, and the absorbance of our unknown concentration (0.317 A) plotted, we estimated a concentration of around 0.84 mg/mL of protein. Our calculations indicated a quantity of 168 mg of protein, which was an approximately 8.96% yield of the projected 1875 mg that was expected. Errors that may have led to this small yield percentage may have stemmed from our previous lab and our initial attempts to extract the desired amount of protein.
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...
Sequence and structural proteomics involve the large scale analysis of protein structure. Comparison among the sequence and structure of the protein enable the identification on the function of newly discovered genes (Proteoconsult, n.d.). It consists of two parallel goals which one of the goals is to determine three-dimensional structures of proteins. Determine the structure of the protein help to modeled many other structures by using computational techniques (Christendat et al., 2000). This approach is useful in phylogenetic distribution of folds and structural features of proteins (Christendat et al., 2000). Nuclear magnetic resonance (NMR) spectroscopy is one of the techniques that provide experimental data for those initiatives. It is best applied to proteins which are smaller than 250 amino acids (Yee et al., 2001). Although it is limited by size constraints and also lengthy data collection and analysis time, it is still recommended as it can deliver strong results. There are two types of NMR which are one-dimensional NMR and two-dimensional NMR. One-dimensional NMR provides enough information for assessing the folding properties of proteins (Rehm, Huber & Holak, 2002). It also helps to identify a mixture of folded and unfolded protein by observing both signal dispersion and prominent peak. Observation in one-dimensional spectrum also obtains information on molecular weight and aggregation of molecule under investigation. In spite of this, two-dimensional NMR are used for screening that reveal structural include binding, properties of proteins. It also provides important information for optimizing conditions for protein constructs that are amenable to structural studies (Rehm et al., 2002). NMR is a powerful tool which it w...
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...
“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 ...
Figure 3 shows the structure of the prepared protein along with the ligand depicted in green colour.
Protein have connection with amino acid to help in functions of: skin, muscle, hair and bones
The covalent structure of a protein is composed of hundreds of individual bonds. Because free rotation is possible around a good portion of these bonds, there are a very high number of possible conformations the protein can assume. However, each protein is responsible for a particular chemical or structural function, signifying that each one has a distinctive three-dimensional configuration. By the early 1900’s, numerous proteins had been crystallized. Because the ordered collection of molecules in a crystal can only form if all of the molecular units are the same, the discovery that proteins could be crystallized proved that even large proteins have distinct chemical structures. This deduction completely transformed the understanding of proteins and their respective functions. It is important to investigate how a series of amino acids in a polypeptide chain is translated into a three-dimensional protein structure. There are five general topics related to this process: the structure of a protein is determined by its amino acid sequence, the role of a protein is dependent on its unique structure, an isolated protein typically exists in a small number of stable forms, non-covalent interactions are the most important stabilizing forces in a protein structure, and there are structural patterns that aid in explaining and understanding protein architecture.
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
“Proteins are large, complex molecules that play many critical roles in the body” (Genetics Home Reference, 2014, p. xx-xx). “They do most of the work in cells and are required for the structure, function, and regulation of the body’s tissues and organs” (Genetics Home Reference, 2014, p. xx-xx). “Proteins are made up of hundreds or thousands of smaller units called amino acids, which are attached to one another in long chains” (Genetics Home Reference , 2014, p. xx-xx). “There are 20 different types of amino acids that can be combined to make a protein” (Genetics Home Reference, 2014, p. xx-xx). “The sequence of amino acids determines each protein’s unique 3-dimensional structure and its specific function” (Genetics Home Reference, 2014, p. xx-xx).
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.
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.
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...
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".