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Protein Folding

This document discusses protein denaturation and renaturation. It defines denaturation as the process where a protein loses its native 3D structure and discusses common denaturants like heat, acids, bases, and detergents. It also summarizes Anfinsen's classic experiment which showed that a protein's primary structure contains all the information needed to refold it into its correct 3D structure after denaturation.

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52 views32 pages

Protein Folding

This document discusses protein denaturation and renaturation. It defines denaturation as the process where a protein loses its native 3D structure and discusses common denaturants like heat, acids, bases, and detergents. It also summarizes Anfinsen's classic experiment which showed that a protein's primary structure contains all the information needed to refold it into its correct 3D structure after denaturation.

Uploaded by

9374 DIVYATA PATIL
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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• Denaturation and Renaturation of proteins;

• Denaturants and their mode of action;


• Anfinsen’s classical experiment;
AMINO ACID
• Denaturation is the process in which protein loses its
native confirmation
• It basically involves the disruption and possible destruction
of both the secondary and tertiary structures
• It mainly breaks the covalent and non covalent bonds, but
they are not strong enough to break peptide bonds
• So the primary structure remains intact
• Precipitation or coagulation or flocculation of protein
occurs
• Heat
• Strong acids
• Strong bases
• Detergents
• Reducing agents
• Heavy metal ions
• Organic solvents
• Solutes
• Most proteins can be denatured by heat, which
affects the weak interactions of the proteins in a
complex manner
• Interactions affected: Hydrogen bond
Nonpolar (Hydrophobic) bond
• As Kinetic energy increases, molecules vibrate
rapidly and violently
• Protonation and Deprotonation of the side groups
of protein occurs
• Interactions affected: Salt bridges,
Hydrogen bond,
Electrostatic interaction.
• Detergents are amphipathic molecules
• Interactions affected: Hydrogen bonds,
Non-Polar bonds (Hydrophobic)
• Disulphide bonds are formed by oxidation of
sulfhydryl groups on cysteine
• Reducing agent reduces the disulphide bonds to
sulfhydryl groups & breaks intra and interchain
disulphide bonds
• Interactions affected: Non-covalent bonds
(Disulphide bonds)
Reducing agent:
Beta- Mercaptoethanol
Urea

Reductants add Hydrogen atoms to make the thiol


group (-SH)
• Heavy metal usually contains: Hg+2, Pb+2 & other
metals with high atomic weights.
• Heavy metal salts denature proteins the way acids
and bases denature.
• Interactions affected: Non-covalent bonds
(Disulphide bonds)
The reaction of a heavy metal salt with a protein
usually leads to an insoluble metal salt protein
• They act primarily by disrupting the hydrophobic
interactions that make up the stable core of
globular proteins
• New hydrogen bonds are formed instead
between new alcohol molecule and protein side
chains
• Interactions affected: Hydrogen bonds,
Polar (Hydrophillic bonds)
• Reversible denaturation is
called Renaturation.
• Example: Hemoglobin
undergoes denaturation in the
presence of salicylate. By
removal of salicylate,
hemoglobin is renatured
• Example: Anfinsen’s
Experiment
• The classic experiment, carried out by Christian
Anfinsen in the 1950s, provided the first evidence
that the amino acid sequence of a polypeptide
chain contains all the information required to fold
the chain into its native, three-dimensional
structure.
• Later experiments proved that the primary
structures determines the conformation of the
protein
• In this following experiment he used a particular
type of enzyme called Ribonuclease
• Urea: Disrupts the non-covalent bonds such as
ionic interactions and hydrogen bonds
• Mercaptoethanol: Breaks down the disulphide
bonds by an oxidation- reduction reaction
• He conducted 3 experiments.
• When the native enzyme was treated with excess
Beta-Mercaptoethanol and 8.0M Urea, all disulfide
bonds and hydrogen bonds were broken and the
protein denatured. When the denaturing agents
were removed the enzyme eventually reformed its
original tertiary structure
• When Beta-Mercaptoethanol was removed first, an
inactive enzyme was formed. This is because the
improper disulfide bonds were formed
• When trace Beta-
Mercaptoethanol was added
to the scrambled enzyme,
the correct tertiary structure
was eventually reformed.

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