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LET'S TALK LIFE-SCIENCE BIOCHEMISTRY

Suraj Prakash Sharma | Ekta Chotia

PROTEIN STABILITY
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6.            "PROTEIN STABILITY"

The major factors affecting the protein stability :

6.1.         Hydrophobic effect :

It is a major factor for the folding of globular protein. In a  protein hydrophobic and hydrophilic both types of amino acid are present. Different hydrophobic side chains of amino acids in the protein molecules come into contact with one another and increase the stability of the system. Water molecules form hydrogen bond with themselves and produce three dimensional cluster. The formation of cluster is result in decrease in the entropy of the system. Maximum stability is achieved by the hydrophobic side chains which are one inside the protein molecule (core region) and hydrophilic group which are out of the surface of protein molecules and have high attraction for water. Due to this structure the entropy of protein become decrease and stability increases.

Order of hydrophobic amino Acids : Phenyl alanine > Alanine  >  > Glycine > Leucine

When the temperature decrease, the strength of the hydrophobic effect also decreases, which is probably the major cause of cold denaturation in protein.

Hydrogen bond : When a hydrogen atom is present between two electronegative atoms, it forms an extra bond (electrostatically interaction). This extra bond known as hydrogen bond. The strength of hydrogen bond (2 to 10 Kcal/mol) contributes in the stability  of a folded protein.

In the unfolded state, all hydrogen bonding members in the extended chain are formed the hydrogen bonds with water. When  protein folds in water all hydrogen bonds are broken, and only some are replaced (only 20%) by intra protein  hydrogen bond.

Salt bridges : Salt bridges is an important factor for stabilizing the native structure of protein. Formation of salt bridges depends on the ionization properties of the amino acids groups.

Salt bridges is actually a combination of two non-covalent interaction.

(i)            Hydrogen bond

(ii)           Electrostatic interaction

The salt bridges mostly arises from the anionic carboxylate (of aspartic acid or glutamic acid) and the cationic ammonium (of lysine or Arginine). The distance must be less than 4Å between the residues which are participating in the salt bridges. If distance is more than 4Å, amino acids can’t form salt bridges.

Aromatic Aromatic Interaction : Most of the aromatic side chain ( 60%) found in proteins are involved in aromatic pairing. The partially positively charged hydrogen of one ring can interact with the partially negatively charged carbon of the other ring.

Metal binding : Many proteins can be stabilize by metal binding because metal ion are coordinated by lone pair (e–) electrons of oxygen or nitrogen atom. Metal ion binding contributes 6 – 9 Kcal/per mol to stability.

Disulphide Bond : Oxidation of two cysteine residue to form a covalent disulphide bond. This bond may be present between same or different proteins molecules. The enthalpy of a covalent S-S bond is very high due to the conformational stability of protein increase. Thus the S-S bond contributes in a great way to stability. If we introduced a disulphide bond in RNase H, the stability of RNase H is increased.

Conformational entropy of protein : The conformational entropy associated with the number of conformation of a molecule. The entropy of denatured protein is significantly high than the native state of protein.

Disulphide bond stabilized the folded form of protein in several ways

  1. Increase the effective local concentration of protein residue and lowers the effective local concentration of water molecules.
  2. Holds two portions of protein together.
  3. Form hydrophobic core of the folded protein.

6.2.         Factors that decrease the stability of protein :

  1. Heavy metal ion : Heavy metals disrupt the salt bridges by forming ionic bond. eg. Mercury.
  2. Detergent : Detergent disrupt the hydrophobic interactions and extended the protein into unfolded polypeptide chain.
  3. High temperature : High temperature disrupt the non-covalent interaction of protein.
  4. Reducing agent : Reducing agents reduces the disulphide bond. eg. Performic Acid, b-mercaptoethanol.
  5. pH : Change in pH result in protonation or deprotonation of amino acids of protein.
  6. Salinity : Ions from the salt bind to side chain (R) group and disrupt the salt bridges.
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