Protein Structure and Denaturation
We have seen how protein structure is assembled into a native conformation. The reverse process can also occur. It is called denaturation and results in the destruction of the native conformation of a protein and so, of its biological function. Protein function depends absolutely on its structure.. In denaturation, the peptide bonds are not affected, but the H-bonds, disulfide bonds, salt bridges and hydrophobic interactions can all be disrupted, leading to the consecutive alteration of 4o, 3o and 2o structure. When there is quaternary structure, it is disrupted first. Next, tertiary structure is disrupted and globular proteins unfold, losing their hydrophobic interior spaces. Finally, H-bonds are broken and random coil results. This process is sometimes reversible and the protein can be renatured.
How are proteins denatured?
1. Heat. If energy is added to a protein, vibrational modes within the molecule will be excited. This will disrupt hydrophobic interactions and dipole-dipole interactions, and denature the protein. Most proteins are denatured above 50oC, a temperature well above the normal body temperature of 37oC. The most common manifestation of this is frying an egg. Burns, even sunburn, cause denaturation of proteins in the skin. Mechanical energy can also be used to denature a protein. Whipping egg whites denatures the proteins.
2. Acids and Bases. When the pH of a solution containing protein is changed, the protonation state of the amino and carboxylate groups changes, and ionic bonds in the proteins will be disrupted. This disrupts salt-bridges If the pH change is small, reversal of the pH change will often regenerate the native structure. Remember too, that changing the pH can lead to the precipitation of proteins. If the pH approaches their isoelectric point (pHI ) at which the protein carries no net charge, the protein will no longer be solvated and will aggregate and precipitate out of solution. The buffering capacity of blood serum is well regulated to prevent precipitation of blood proteins by preventing low pH formation.
3. Detergents. Long alkane chains, like in sodium dodecyl sulfate,
tend to unfold globular proteins, exposing buried portions to the outside. The detergents then interact with the non-polar R-groups via London Dispersion Forces preventing the R-groups from interacting with each other.
4. Oxidizing and Reducing Agents. Reducing agents can break disulfide bonds, leading to a loss of structure. Oxidizing agents can create new disulfide bonds where they don't belong. This is the process used in hair "permanents". A reducing agent is put on the hair to break existing disulfide bonds. The hair is then arranged in a new conformation (curlers) and an oxidizing agent is added to form new disulfide bonds to maintain this new structure.
5. Hydrogen bonding organic solvents. Alcohols and ethers, when added to protein solutions, disrupt H-bonds within the protein molecule. Ethanol, for example, passes through the cell wall of bacteria and denatures bacterial proteins.
6. Urea and guanidine.
These are called chaotropes and they function by disrupting the H-bonding of water at the surface of the protein. Normally, globular proteins have a sort of water "cage" around them, water molecules H-bonded to each other, surrounding the protein. When the water H-bonds to the chaotropes instead, the protein is more likely to open up and expose the protein interior. The salts of these chaotropes can usually be removed from solutions, using dialysis, and the protein can usually be renatured.
7. Heavy metal salts. Heavy metal salts, Ag+, Hg+ and Pb+ denature proteins by reacting with the sulfhydryl groups to form stable, metal-sulfur bonds. This prevents formation of needed disulfide bonds. Metal ions can also combine with the carboxylate ion on R-groups, preventing their participation in salt bridges.