Molecular & Cell Biology Protein Folding and Misfoldingoscar.iitb.ac.in/onsiteDocumentsDirectory/Protein Folding and... · Molecular & Cell Biology. 3. Creutzfeldt–Jakob disease:

Folding of proteins into their native conformations occurs spontaneously under physiological conditions and is dictated by the primary structure of the protein.

Protein Folding and Misfolding

After interacting with this Learning Object, the learner will be able to, ØDescribe the Thermodynamics of protein folding.ØDescribe Anfinsen’s experiment.ØDescribe Amino acid structure determines 3-D foldingØList out Molecular chaperones for protein foldingØRecall Protein misfolding diseases.

Learning Objective

Protein Folding and MisfoldingMolecular & Cell Biology

An unfolded polypeptide chain has very high free energy and entropy. Protein folding acts to decrease the free energy of the system by forming favorable interactions and assuming a more stable state. The entropy of the polypeptide chain decreases during this process.

Thermodynamics of protein folding


As the protein continues to fold in order to assume its stable, low energy native state conformation, the entropy also decreases. While this would seem unfavorable for the system, it must be recalled that the entropy of the surrounding water molecules increases during the process, thereby increasing the overall entropy and making it favorable and spontaneous.



Ribonuclease A in its native state has four disulphide bonds between its cysteine residues. When treated with b-mercaptoethanol and 6M urea, the protein undergoes denaturation and the disulphide linkages are broken. Enzyme activity is lost in the denatured state.

Anfinsen’s experiment


It was observed by Anfinsen that removal of urea and -mercaptoethanol led to the refolding of the enzyme to assume its native state with more than 90% enzyme activity being intact. However, if only

-mercaptoethanol was removed in presence of urea, the formation of disulphide bonds was random, leading to enzyme with only around 1% activity.



The process of protein folding is governed by the distribution of polar and non-polar amino acid residues in the protein. Hydrophobic amino acids are driven to interact with one another, a process termed as hydrophobic collapse. They come together andin the process, eliminate water molecules around them. The polar residues remain on the surface and form hydrogen bonds with water molecules while the hydrophobic residues get buried within the core of the protein.

Amino acid structure determines 3-D folding


Proteins typically adopt only one characteristic functional native state conformation which has lowest free energy and is most stable. Folding is limited to one conformation due to properties of the amino acid side chains such as hydrophobicity, size, shape etc. Folding is a highly cooperative process wherein there is progressive stabilization of the intermediates. Although it is theoretically possible to predict protein structure from the amino acid sequence, several long-range interactions often limit these predictions.



The unfolded protein is bound by DnaJ and then by DnaK which is an ATP bound protein. The hydrolysis of ATP into ADP and Pi by DnaK is stimulated by DnaJ. The resulting DnaK-ADP remains tightly bound to the unfolded protein.

Molecular chaperones for protein folding


The nucleotide exchange factor GrpE present in bacteria facilitates release of ADP along with DnaJ. This leaves the DnaK bound to the partially folded protein which continues to undergo folding to a more favorable low energy conformation.



Once the protein gets completely folded, it gets detached from DnaK which then binds ATP again, thereby completing the cycle and preparing it for the next round of protein folding. Any protein which may not have been folded completely is then taken over by the GroEL chaperonin system which completes the folding.



Protein folding & misfoldingMolecular & Cell Biology


1. Entropy: Entropy is a measure of randomness or how disorganized a system is and forms the basis of the second law of thermodynamics, which states that the total entropy of a system cannot decrease without correspondingly increasing the entropy of another system. In other words, the entropy of the universe (system + surrounding) is constantly increasing. Entropy helps in predicting the spontaneity of any process. An unfolded polypeptide chain has high entropy which goes on decreasing as the protein folds into its native state.

2. Free energy: The free energy, also known as Gibbs free energy, is the maximum amount of mechanical work that can be done by a system at constant temperature and pressure. In general, all systems try to attain minimum free energy and a reaction takes place spontaneously only when the associated free energy change is negative.

3. Unfolded polypeptide chain: The amino acids that have been joined together by peptide bonds but have not yet formed their secondary or tertiary structures. This conformation has the highest free energy and entropy.

4. Partially folded polypeptide: The amino acids in the polypeptide chain start interacting by means of hydrogen bonds across the polypeptide backbone in order to initiate the folding process. The free energy and entropy of the system gradually decrease as the folding takes place.


5. Native state α-helix: The polypeptide chain assumes its most stable, native conformation in the form of an α-helix with the folding being directed largely by its amino acid residues. This conformation corresponds to minimum free energy and entropy, thereby conferring very high stability. The lowering of entropy is favoured by a corresponding increase in entropy in the surroundings composed of water molecules.

6. Molten globule: Initial collapsed state of a protein with very little thermodynamic stability is known as the molten globule. The amino acid side chains are extremely disordered in this state with several fluctuations being observed.

7. Percentage residues in native conformation: This refers to the number of residues that have assumed their favourable, lowest energy states. The percentage increases gradually as the folding process takes place.




3. 6M urea: It is an organic compound having two amine groups joined by a carbonyl group and used at concentrations up to 10 M for denaturing proteins by breaking the noncovalent interactions.

4. Denatured ribonuclease A: On treatment with β-mercaptoethanol and urea, the ribonuclease A loses its native conformation due to breaking of the disulphide and noncovalent linkages. Activity of the enzyme is also lost during this process. However, it was observed by Anfinsen that removal of both urea and β-mercaptoethanol allows the enzyme to fold into its native conformation again with more than 90% enzymatic activity.place.

1. Native ribonuclease A: This is an endonuclease enzyme composed of 124 amino acids that cleaves single-stranded RNA molecules. It has four disulphide bonds in its native state that are essential for conformational folding and enzymatic activity. This was used by Christian Anfinsen to postulate the thermodynamic hypothesis of protein folding, according to which the folded form of a protein represents its free energy minimum.

2. β-mercaptoethanol: β or 2-mercaptoethanol with the formula OHCH2CH2SH is a chemical compound that is used commonly to reduce disulphide linkages in proteins, thereby disrupting the tertiary and quaternary structures.


1. Amino acid sequence 1, 2: These are two completely different amino acid sequences that will give rise to different protein structures.

2. Protein 1, 2: The protein structure corresponding to amino acid sequence 1 and 2 respectively. The first amino acid sequence cannot give rise to the second protein structure & vice versa.

3. Protein folding: The process by which the amino acid side chains in the proteins interact with one another to form energetically favourable bonds with each other thereby allowing regions that are far away from one another to move closer. This process is determined by the amino acid sequence of the proteins and needs to be energetically feasible in order to take place.




1. Unfolded protein: This refers to the protein or polypeptide chain that has not been folded or is in a partially folded state.

2. DnaJ and Dna K: These are molecular chaperones found in E.coli that are analogous to the eukaryotic heat shock protein (Hsp) chaperone system. These chaperones are proteins that interact with unfolded or partially folded proteins and provide them with suitable microenvironments in which folding can occur. In addition to this chaperone system, the Hsp proteins have also been studied and have been found in abundance in cells that have been stressed by elevated temperatures.

3. ATP: Adenosine triphosphate (ATP) is the energy currency of the cell due to its high energy phosphate bonds. It gets hydrolyzed to liberate adenosine diphosphate (ADP) and a phosphate group (Pi).

4. GrpE: This is a nucleotide exchange factor present in bacterial systems that facilitates the release of bound ADP.

5. GroEL system: The GroEL system refers to another group of elaborate protein complexes known as chaperonins that assist the folding of several cellular proteins.


1. Alzheimer’s Disease: a) Structure of certain normal soluble cellular proteins normally rich in alpha helical regions converted into beta strand conformations which further link with each other to form beta sheet aggregates known as amyloids. b) Insoluble amyloid plaques are essentially made up of a single polypeptide chain or fibrils known as amyloid-β-protein (Aβ).c) Observed in the brain of patients with Alzheimer’s where dead or dying neurons surround plaques. d) Neurotoxicity believed to be caused by the Aβ fibrils before they get deposited as amyloid plaques. e) The disease presents various symptoms such as memory loss, decreased neuromuscular coordination, confusion and dementia.

Protein misfolding diseases

2. Huntington’s disease: a) Neurodegenarative disorder of genetic Origin

affecting muscular coordination.b) Caused by increased number of trinucleotide repeats, CAG, in Huntingtin gene leading to increased number of glutamine residues incorporated in corresponding protein.c) This alters the folding of the Huntington protein which has highest concentration in brain and testes. d) Exact function of the protein is unclear but is known to interact with several other proteins. e) Mutated protein has also been found to have effects on chaperone proteins which in turn help in folding several other proteins. f) Prominatly affects basal gauglia which plays a key role in movement and behavioural control.


3. Creutzfeldt–Jakob disease: a) Initially believed to be caused by viruses or bacteria. b) Later discovered to be transmitted by small proteins known as prions. c) Prion proteins composed of beta sheet structures that have been modified from previously existing alpha helices. d) Protein aggregates of one abnormal protein sufficient to function as a nuclei for other normal proteins to attach themselves to. e) Characterized by muscular spasms, loss of muscle control and memory loss.


4. Cystic fibrosis: a) Autosomal recessive disorder caused by a mutation in gene for the protein cystic fibrosis transmembrane conductance regulator (CFTR) .b) CFTR regulates components of sweat, digestive juices and mucus. c) Dysfunctional protein gets degraded by the cell.


d) Disorder can affect several body parts such as the lungs, GI tract and reproductive organs.

5. Pulmonary emphysema: a) Progressive disease of the lung causing shortness of breath. b) Can be caused by deficiency of the protein alpha-1-antitrypsin (A1AT).c) A1AT is responsible for protecting the lung tissues from damage by enzyme neutrophil elastase. d) Abnormally secreted A1AT gets accumulated in the liver thereby allowing lung tissue damage.e) Causes wheezing,shortness of breath, asthma-like symptoms and also liver cirrhosis.

6. Lathyrism: a) Regular ingestion of seeds from sweet pea (Lathyrus odoratus) causes disruption of cross-linking in the muscle protein, collagen. b) Collagen is an important structural protein having a triple helical structure.


c) Cross-links formed are due to the oxidation of lysine residues by the enzyme lysyl oxidase to form allysine. d) These are essential for proper folding of collagen, giving it the required strength. e) β-aminopropionitrile, present in abundance in sweet pea, deactivates this enzyme by binding to its active site f) This prevents cross-linking and proper folding of the protein.g) Causes muscle fragility and weakness.
