Anfinsen experiments indicated that the formation of higher-order structure mainly depends on nature and type of amino acids present in the primary structure.
In his experiment, he has performed denaturation and renaturation process using pancreatic ribonuclease as the experimental protein. RNAse is an extracellular protein having a single subunit containing 8 cysteine residues that can be organized to form four disulfide bridges. The tertiary structure of RNase is stabilized by both covalent and noncovalent interactions. He measured the biological activity of the native RNase before and then subjected the enzyme to denaturation using the β-mercaptoethanol and urea as denaturing agents. The denaturation process removes the biological activity of the enzyme. When anfinsen renatured the denatured proteins by using the method dialysis, interestingly he found the regaining of biological activity in previously denatured proteins. The process of dialysis removes the all low molecular weight denaturing compounds. The main intention of his experiment is to prove that the information in an amino acid sequence of a protein is important for protein folding during tertiary structure formation. Some exceptions of this experiments are all proteins cannot go spontaneous refolding as like RNase enzyme.
For some of the proteins, the folding will be completed by chaperons which are a group of Heat shock proteins (HSP) proteins that facilitate the protein folding as well as subunit-subunit interaction. One of the mechanisms of chaperons is facilitate the protein folding by bringing hydrophobic side chains together and form nucleation center.
Thermodynamically spontaneous process in protein folding indicates the changes in negative free energy [∆G<0].
∆G for a process depends on ∆H, ∆S and temperature [T]
∆G= Free energy change
∆H= enthalpy change
∆S= entropy change
Protein folding is a biological process in the living cell, that does not depend on the temperature.
So, In the process of protein folding there is no change in temperature, since ∆H and ∆S will be the major contributors to the free energy change.
The entropy change has two components
1) Change in the entropy of the protein
2) Change in the arrangement of water molecule present surrounding to protein.
If protein folding converts highly randomly coiled proteins to highly organized compact form then the entropy will decrease and therefore it will have a negative contribution to the folding.
In the case of the water molecule, the protein folding increases the randomness of well-organized water molecules which was surrounded the randomly coiled protein. Disruption of the well-organized water molecule is required for protein folding takes place. Hence the disruption of water molecules will have a positive contribution to protein folding. Thus the overall entropy change will be negligible and it will not contribute negative free energy change. Hence changes in enthalpy of the protein [∆H] will be the major contributor to the negative free energy change.
As the protein folding occurs the energy content of the protein decreases and therefore ∆H become negative which in turn makes ∆G negative and folding become spontaneous.