Destruction and building of the secondary and tertiary

structures of proteins: Denaturation and folding of proteins

Proteins are synthesized on ribosomes as linear polypeptites.

As they are synthesized they assume secondary and tertiary structure.

Activity of proteins depend on the integrity of its final tertiary
structure also reffered as the native form.

The native form of protein is not very stable structure and it

vulnerable to change by heat, high salt, reducing agents,detergents or
organic solvents. All these treatments destabilize the week
interactions responsible for the higher order structures.

Destruction of the higher order structure is leads to the loss of activity

of a protein. This process is referred as denaturation or unfolding of
protein.
Denaturation may not require complete unfolding of proteins. It
might be still a folded structure but in random conformation.

Denaturation is cooperative, I.e. changes in one part of protein acelerate

the unfolding of the other part.

Some proteins are resistant to denaturation by heat (Proteins of

hot spring bacteria stable at 100 oC). The primary structure of
these proteins are not very different from those from normal
bacterium. It remains a biochemical puzzle to explain the stability
these proteins.

Heat: destabilizes H-bonding

Detergents, Urea, organic solvents: destabilize hydrophobic
interactions
Extreme pH conditions: cause ionization of side chains resulting
in electrostatic repulsion and collapse of structure.
Denaturation of proteins by heat and guanidine hydrochloride
Amino acid sequence determines tertiary structure:

Christian Anfinsen, 1950: Denaturation of ribonuclease by urea and

reducing agent led to complete loss of activity.

Removing the urea and reducing agent from this mixture resulted in
complete renaturation of this enzyme.

8 cys residues: four disulfide bonds

Mathematically there could be 105 ways to make these bonds. But only
one way of the native peptide is assumed after renaturation.

Chemically synthesized enzyme used in this study also showed similar

results ruling out any role of a contaminants in renaturing process.
Renaturation of
Ribonuclease
Folding of polypeptide is rapid in a stepwise fashion:

1. Proteins are synthesized and folded in different confomation and

the one of which matching with native conformation is selected
(trial and error method). Not possible

2. Proteins keep folding in the correct conformation as it is

synthesized in a step wise fashion.

Initially the unfolded peptide has highest randomness (entropy) and as

it keeps assuming more folded structure the entropy keeps
decreasing.
A computer simulated pathway of folding of villin protein
(36AA long polypeptide)
Assisted folding of proteins:

Not all proteins fold spontaneously as they are synthesized in

the cells. Folding of many proteins is facilitated by a specialized
class of proteins called Chaperones.

Class I: HSP70 type chaperones: HSP70, HSP40, DnaK, DnaJ

Class II: Chperonines: GroEL, GroES

Other enzymes involved in protein folding:

Protein disulfide Isomerase (PDI)

Peptide Prolyl cis-trans Isomerase (PPI)

Diseases caused by the defect in protein folding:

Cystic fibrosis: Defect in the folding of cystic fibrosis Tran membrane

conductance regulator protein.

Diseases caused by misfolding of Prion proteins:

Kuru Disease
Creutzfedlt-Jakob Disease
Scrapie Disease in sheep
Mad cow disease

Misfolded prion protein act as infectious agents.

They act as chaperons which can multiply by binding to normal PrP

and folding it to dangerous form similar to itself.

Mechanisms of the functions of normal prions and the dangerous ones

are still not clear.
Stained section from the cerebral cortex from a patient with
Creutzfedlt-Jakob disease indicating spongiform patahlogy
PrP (harmless)

PrPsc (infectious)
Hypothetical mechanism of action of PrPSc protein