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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.
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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-bondingDetergents, Urea, organic solvents:
destabilize hydrophobic interactionsExtreme pH conditions: cause
ionization of side chains resulting in electrostatic repulsion and
collapse of structure.
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Denaturation of proteins by heat and guanidine hydrochloride
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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 bondsMathematically 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.
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Renaturation of Ribonuclease
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Folding of polypeptide is rapid in a stepwise fashion:
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
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.
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A computer simulated pathway of folding of villin protein (36AA
long polypeptide)
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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)
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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 DiseaseCreutzfedlt-Jakob DiseaseScrapie Disease in sheepMad
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.
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Stained section from the cerebral cortex from a patient with
Creutzfedlt-Jakob disease indicating spongiform patahlogy
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Hypothetical mechanism of action of PrPSc protein