Proteomics/Protein - Protein Interactions/Binding Sites

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« Protein Interactions
Characterization of Binding Sites
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Types of Interfaces Allosteric Effects and Binding Affinities

Page Edited and Updated by: Christopher Fucile
E-mail: cff8255@rit.edu


This Section:


Characterization of Binding Sites[edit]

Protein stability and flexibility are dependent on electrostatic interactions such as hydrogen bonding and van der Waals forces. The energy of the interaction is not evenly distributed between the two proteins at the interface. Rigidity in the interface ensures that the entropy loss is offset and the binding free energy is contributed to in a favorable way by the amino acid residues contained in the interface. Contributions of hot spots to the stability of the protein-protein complex within a hot region is cooperative, however the contributions of independent hot regions are additive.

  • Hydrogen Bonding/Hydrophobic Packing
  • Dehydron Bonds
  • Salt Bridges
  • van der Waals
  • Cation-π Interactions
  • Pockets
  • Hot Spots
  • Protein Chips

Hydrogen Bonding and Hydrophobic Packing[edit]

Hydration is very important for the three dimensional structure and activity of proteins. Surface water molecules are held most strongly by the positively charged basic amino acids. Hydrogen bonding adds conformational flexibility, while the level of hydration determines the degree of flexibility in the internal molecular motions. Water molecules in close proximity to peptide hydrogen bonds cause separation in the bonds and a release of tension in the structure. The presence of water molecules also prevents swift conformational fluctuations from occurring. Water interacts with protein surfaces; the breaking of hydrogen bonds between the water molecules and the amino acids has an unfavorable enthalpic result on the system, effecting the overall energy of the protein. Water molecules act as an adhesive, sealing holes in complementary surfaces that would normally lack shape. Hydrogen bonding drastically reinforces other bonding present in the interface between proteins. This type of bonding occurs when an hydrogen atom proximal to a docking facade interacts across the interface with atoms of another molecule. Hydrogen bonding can occur between side chains, backbone groups, and between the two.[1]

Hydrophobic surfaces located at ligand receptor interfaces have greater ambiguity with bonding. Hydrogen bonding between the peptide atoms decreases the hydrophobicity of the backbone. Coincidentally, hydrophobic surfaces can be sealed with an ordered shell of water molecules, creating an entropically favorable state when water molecules are ejected. Water removal increases the thermodynamic benefit in hydrophobic patches. These patches are uncommon though; the patches found in proteins are more often hydrophilic. The secondary structure of the protein is a result of hydrogen bonding since the residues arrange themselves in such a way so that hydrophobicity of the backbone is reduced. α-helices and β-sheet conformations are the way in which is accomplished.[2]

Amino acids in order of hydrophobicity[edit]

Least hydrophobic to most hydrophobic:

  • Lysine
  • Glutamic Acid, Aspartic Acid
  • Arginine, Glutamine, Serine, Aspargine
  • Proline, Glycine, Threonine
  • Histidine, Alanine
  • Tyrosine
  • Cystine, Valine, Tryptophan, Methionine
  • Leucine, Isoleucine, Phenylalanine

Dehydron Bonding[edit]

The hydrogen bonds in the backbone of a protein usually exist in the form of dehydron bonds, which form between two hydrogen atoms bonded that differ in polarity bonds. The proton acceptor in this case is a metal hydride with the donor being OH or NH. Dehydrons attract hydrophobes, their bonding increases the electrostaticity of hydrogen bonds by increases the surrounding dielectric coeffiecient. The dehydron bonds thus create a hydrophobic shell around the backbone increasing the electorstatic interaction; this desolvated environment is condusive for salt bridge formation increasing structural stability.[3]

Salt Bridges[edit]

Salt bridges, also referred to as ionic bonds, are a form of chemical bonding that occur between amino acids of opposite charge. They contribute greatly to the stability of the protein and are usually found in the interior of the protein structure. This stability is dependant on the location of the side chains, and the electrostatic contribution. Hydrophobic environment is conducive to salt bridges. Charged residues within four angstroms to each other have the ability to form salt bridges.[4]

Van der Waals Interactions[edit]

Van der Waal forces occur between dipoles. Covalently bonded atoms differ in electronegativity that causes a minute dipole. Since the distribution of electrons is asymmetrical, a dipole effect is created by the van der Waals attraction. These dipoles oscillate, creating a dipole field. This field allows two atoms with identical oscillation frequencies to synchronize, one negative while the other positive. Thus the charge between the two cancel each other out and the net charge is zero. van der Waals bonds are one of the weakest intermolecule forces studied. But van der Waal bonds are important, since it is with these forces that noble gases can achieve bond saturation with the dominant form of interaction between electrically neutral species. Induced dipole interactions cause van der Waal bonding to occur, and the large close surface contact allows van der Waal binding to contribute significantly.[5] The following formula is used to determine van der Waal bonding:

A/(r6) - B/(r12) where r is the distance between A and B.

Pockets[edit]

A protein pocket is a concave surface region containing the active sites of the protein. These areas are solvent accessible, and are characterized as either unfilled or complemented, the partner conformation not being taken under consideration. Compositional difference between complemented pockets and other surface pockets may be useful for predicting complemented pockets from the unbound states. The floor of complemented pockets are often filled with residues that are ionizable and/or polar. In a hydrophobic environment, these residues provide molecules for salt bridge formation and hydrogen bonding which are essential for binding stability. Pockets are crucial for binding site flexibility for large interfaces reduce binding stability, and allow for ligand movement.

Cation-π Interactions[edit]

Cation-π pairs are frequently present in the interface of proteins, probably due to their low energy conformation. They play an important role in specificity, causing a need for conformational changes to provide the correct orientation for binding. Cation-π interactions are found most frequently in homodimers. The abundance of this form of bonding is tied to the occurrence of specific amino acids. In homodimers, arginine bound to phenylalanine forms the most frequent cation-π bonds, while in the other protein complexes arginine-tyrosine and arginine-tryptophan cation-π bonding is a more likely occurrence.

Hot Spots[edit]

A hot spot is an area of high energy and binding around an amino acid residue. Surrounded by hydrophobic pockets, hot spots are found in clusters, rather than scattered throughout the interface. This clustering assists with the elimination of water molecules, and increases the bond strength and available bond energy through increases charge-charge relations. Hot spots usually do not include hydrogen bonding, electrostatic interactions, although there is a slight preference of non-polar residues. Packing around hot spots is significantly tighter that the other residues found in the interface. Since hot spots contribute significantly to binding, the association of neighboring residue size, charge, and interaction must be taken under consideration when considering binding affinity and docking. It has been established that a linear relationship between the number of hot spots and the interface size exists; the larger the interface the higher the number of hot spots present.

Protein Chips[edit]

Much like DNA microarrays, and gene chips, protein chips have revolutionized the method and speed with which scientists can discover differences in gene expression levels, protein chips are a relatively new addition to proteomics that brings high-throughput efficiency to discovering protein-protein interactions. Earlier protein chips emulated existing proteomic techniques by requiring labeling of proteins, a requirement that resulted in only revealing certain proteins or classes of proteins. Recent developments have put forth label-free techniques that couple the original protein chip idea with MALDI-TOF mass spectrometry, and other forms of mass spectrometry, to produce a protein chip technology that does not require proteins to be labeled, consequently widening the range of protein-protein interactions detected with each chip.[6][7] While protein chip technology does not directly contribute to binding site characterization, it is a useful method for gaining insight into how proteins and classes of proteins function through their interactions with many other proteins. See chapter on protein chips more more information.

References(Open Access)[edit]

1.^ "Hydrogen bond" Wikipedia, The Free Encyclopedia. Wikimedia Foundation, Inc. 30 March 2008. <http://en.wikipedia.org/wiki/Hydrogen_bonding>.

2.^ "Dehydron" Wikipedia, The Free Encyclopedia. Wikimedia Foundation, Inc. 30 March 2008. <http://en.wikipedia.org/wiki/Dehydron>.

3.^ "Van der Waals force" Wikipedia, The Free Encyclopedia. Wikimedia Foundation, Inc. 30 March 2008. <http://en.wikipedia.org/wiki/Van_der_Waals_force>.

4.^ "Proteomics/Protein Identification - Mass Spectrometry" Wikibooks. 30 March 2008. <http://en.wikibooks.org/wiki/Proteomics/Protein_Identification_-_Mass_Spectrometry>.