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Protein Interactions Detection

Proteins are the fundamental components of living cells involved with complex biological processes, such as nutrition transport, immune responding, tissue contraction, and gene expression. Most proteins function through interacting with other biomolecules, such as peptides, nucleic acid, lipoprotein, proteins, etc. (Table 1). The nature of these interactions or bindings is typically reversible so that a cell can rapidly respond to changes in its metabolic and external environment. Although reversible, these interactions are specific such that a protein can recognize and bind their specific ligands from thousands of different molecules surrounded it.

Protein Ligand Function
insulin receptor
nicotinic receptor
cytochrome c
cytochrome c oxidase
Electron transport

Table 1.Biological Functions of Protein-ligand Interactions.A variety of protein and ligand types presented with a diverse sets of functions.

A molecule that binds reversibly to the protein, or receptor, is known as a ligand, which may be any type of molecule including another protein, a peptide, DNA, RNA, a small organic molecule, or a metal ion. Proteins and ligands are usually complementary in size, shape, charge, hydrophobicity, and hydrophilicity. These ligands bind to proteins through non-covalent interactions, which are critical in biology. The site on the protein where the ligand binds is called the binding site. However, one particular protein may have several binding sites each corresponding to a different ligand.

To fully describe a biologically relevant protein-ligand interaction, there are many pieces of information that must be known, including:

  • Stoichiometry: number of ligands binding to the protein,
  • Binding constant: affinity of the ligand for the receptor,
  • Intermolecular forces: electrostatic, hydrophobic, H-bonds, etc.,
  • Structure: spatial structure of the protein-ligand complex,
  • In vivo Data: function of the protein-ligand interaction in a cell,
  • Pathway: surrounding cellular and molecular events.

Each of the above parameters contributes to completely describing the distinct protein-ligand pair and its function in biology. These parameters can be obtained by several techniques.

Experimental Methods for Studying Protein Interactions

Biophysical techniques

Biophysical detection techniques (such as spectroscopy, chromatography, thermal analysis, or electrophoresis) are used to collect structural data and equilibrium constants for a specific binding event of interest. KD values of most biological interactions generally range from 10-3 to 10-15 M with most binding interactions on the micro- to nanomolar scale. Based on the value of the dissociation constant and the required information, we can choose the most suitable research technique. For example, X-ray and NMR provide unparalleled structural data but require high concentrations of substrates, which are not suitable for proteins with limited solubility or low yield, and results can be difficult to achieve. However, fluorescence techniques can be performed with excellent quantitative binding data at very low concentrations (nanomolar). Table 2 provides some common bioresearch techniques and the specific physical properties that they depend on.
Method Physical Property
Gel filtration
Optical rotation
Isothermal titration calorimetry
Size, Charge
Electronic spin
Electronic transitions
Size, Shape, Charge
Nuclear spin
Size, Shape
Index of refraction
Size, Shape, Density
Crystallization structure
Reaction/binding heat

Table 2. Biophysical Experimental Techniques. There are wide ranges of methods to study the binding of ligands to macromolecular receptors like proteins based on a number of different physical properties.

In order to determine the association constant, the value of the free peptide concentration can be measured or a change in the biological or chemical properties of the bound ligand, the bound receptor, or the complex must be detected. Dialysis or chromatographic techniques are often used to detect the concentration of the ligand, whereas spectroscopic techniques can measure a change in some property of the components of the system (such as bound ligand, bound receptor, or complex). In addition, it is also possible to perform competitive assays in which the equilibrium constant is deduced for one ligand binding to the protein when it competes with a ligand of known binding affinity.
Using these experimental results, the protein-ligand binding can be described quantitatively by the equilibrium constant. K. Data can also be analyzed to determine the number of ligand binding sites. By measuring the dependence of K on ionic strength, pH, and other variables, more can be learned about the specific intermolecular forces involved in the binding interface.

Y2H system

Y2H system is a common technique to confirm the in vivo interactions between proteins in living organism utilizing specific yeast. The identification of protein-protein interactions using the Y2H system work optimally for the discovery of intracellular binding partners, but are not appropriate for products of the secretory pathway that include ligands, their binding proteins and cognate receptors. The overall success of the Y2H systems has spawned several variations including the bacterial and mammalian two-hybrid systems. These systems have proven worthwhile due to increased through-put and proper post-translational modification, respectively.

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