The document discusses protein folding and denaturation. It begins by explaining that protein folding is the process by which a polypeptide chain folds into its functional three-dimensional structure. It is driven by hydrophobic interactions, hydrogen bonding, and other forces. The document then describes the four levels of protein structure - primary, secondary, tertiary, and quaternary. It also lists factors that can affect folding and experimental techniques used to study folding. Next, it defines protein denaturation as the loss of native structure, discusses examples, and explains how denaturation occurs and its consequences. Finally, it lists chemical and physical agents that can cause denaturation.

2. Learning Objectives
Protein Folding Protein Denaturation
 What is protein folding
 Importance
 Process of protein folding
 Driving forces of protein
folding
 Factors affecting protein
folding
 Experimental techniques
for studying protein folding
 What is protein denaturation
 Examples
 What happens when protein
denature
 How denaturation occurs at
levels of protein structure
 Consequences of protein
denaturation
 Protein denaturants

3. Protein folding is a process by which a polypeptide
chain folds to become a biologically active protein in
its native 3D structure.

4. Each protein exists as an unfolded polypeptide or random coil
when translated from a sequence of mRNA to a linear chain of
amino acids
This polypeptide lacks any stable (long-lasting) three-
dimensional structure
The newly created protein strand then undergoes
posttranslational modification, in which additional atoms or
molecules are added.
Once this post-translational modification process has been
completed, the protein begins to fold (sometimes
spontaneously and sometimes with enzymatic assistance)
The final shape of a protein determines how it interacts with
its environment.

5. Protein folding occurs in a cellular compartment called
the endoplasmic reticulum. This is a vital cellular
process because proteins must be correctly folded into
specific, three-dimensional shapes in order to function
correctly. Unfolded or miss folded proteins contribute
to the pathology of many diseases.

6.  The folding of a protein is a complex process, involving four
stages, that gives rise to various 3D protein structures
essential for diverse functions in the human body.
 The structure of a protein is hierarchically arranged, from
a primary to quaternary structure.
 The wide variation in amino acid sequences accounts for
the different conformations in protein structure
 To understand how a protein gets its final shape or
conformation, we need to understand the four levels of
protein structure: primary, secondary, tertiary, and
quaternary.

7.  The primary structure of a protein, its linear amino-acid sequence,
determines its native conformation.
 The specific amino acid residues and their position in the polypeptide
chain are the determining factors for which portions of the protein fold
closely together and form its three-dimensional conformation.

8.  Formation of a secondary structure is the first step in the folding
process that a protein takes to assume its native structure.
 Secondary structure is generated by formation of hydrogen bonds
between atoms in the polypeptide backbone, which folds the chains
into either alpha helices or beta-sheets.

9.  Tertiary structure is formed by the folding of the secondary
structure sheets or helices into one another.
 The tertiary structure of protein is the geometric shape of the
protein. It usually has a polypeptide chain as a backbone, with
one or more secondary structures.
 The tertiary structure is determined by the interactions and
bonding of the amino acid side chains in the protein.

10. Quaternary
Structure
Quaternary structure results from folded amino-acid
chains in tertiary structures interacting further with
each other to give rise to a functional protein such as
hemoglobin or DNA polymerase.

12. Folding is a spontaneous process that is mainly guided by :
 Hydrophobic interactions (important driving force
behind protein folding)
 Formation of intra molecular hydrogen bonds
 Van der Waals interactions
 Chaperones ( Protein molecules inside the cell that help
in protein folding )

13. Factors affecting protein folding
Protein folding is a very sensitive process that is
influenced by several external factors including:
 Electric and Magnetic
fields
 Temperature
 pH
 Chemicals
 Space limitation

14. A protein is considered to be misfolded if it cannot achieve its
normal native state. This can be due to mutations in the amino
acid sequence or a disruption of the normal folding process by
external factors
 Misfolded proteins denature easily and lose their structure and
function.
 Incorrect protein folding can lead to many human diseases
such as Alzheimer's disease, Huntington's disease ,Parkinson's
disease and Cystic fibrosis

15.  X-ray crystallography
 Fluorescence
spectroscopy
 Circular dichroism
 Protein nuclear
magnetic resonance
spectroscopy
 Biotin painting
 Studies of folding with
high time resolution
 Proteolysis
 Single-molecule force
spectroscopy
 Dual polarization
interferometery

16.  Denaturation is a process in which a protein loses
its native shape due to the disruption of weak
chemical bonds and interactions, thereby becoming
biologically inactive.

17.  When food is cooked, some of its proteins
become denatured. This is why boiled eggs
become hard and cooked meat becomes firm.

18.  When proteins denature, the cells go through a series of changes, this
results in disruption of cell activity and possibly cell death.
 In case of proteins :
 • A loss of three-dimensional structure, sufficient to cause loss of
function
 ⟰ Loss of secondary, tertiary and quaternary structure of proteins.
 ⟰ Change in physical, chemical and biological properties of protein
molecules.

19. How denaturation occurs at levels of protein structure
 In quaternary structure of protein, during protein
denaturation subunits of proteins are dissociated from each
other and get separate
 Tertiary structure denaturation involves the disruption of:
 ⟰ Covalent interactions between amino acid side-chains (such
as disulfide bridges between cysteine groups)
 ⟰ Non-covalent dipole-dipole interactions between polar
amino acid side-chains (and the surrounding solvent)
 ⟰ Van der Waals (induced dipole) interactions between
nonpolar amino acid side-chains.

20. ⟰ In secondary structure denaturation,
proteins lose all regular repeating patterns such
as alpha-helices and beta-pleated sheets, and
adopt a random coil configuration.
⟰ Primary structure, such as the sequence
of amino acids held together by covalent
peptide bonds, is not disrupted by denaturation

22.  ⧗ The native helical structure of protein is lost
 ⧗ The primary structure of a protein with peptide
linkages remains intact i.e., peptide bonds are not
hydrolyzed.
 ⧗ The protein loses its biological activity.
 ⧗ Denatured protein becomes insoluble in the
solvent in which it was originally soluble.

23. ⧗ Denatured protein is more easily digested. This is due to
increased exposure of peptide bonds to enzymes Cooking causes
protein denaturation and, therefore, cooked food (protein) is more
easily digested.
⧗ Denaturation is usually irreversible. For instance, omelet can
be prepared from an egg (protein-albumin) but the reversal is not
possible.
⧗ Denaturation is associated with increase in ionizable and
sulfhydryl groups of protein. This is due to loss of hydrogen and
disulfide bonds.

24. ⧗ Careful denaturation is sometimes reversible (known
as renaturation). For example - Hemoglobin
undergoes denaturation in the presence of salicylate. By
removal of salicylate, hemoglobin is renatured.

25. Chemical Agents Physical Agents
 Acids and Alkalies
 Organic solvents
 Salts of heavy metals
 Chaotropic agents
 Detergents
 Altered pH
 Heat
 Violent shaking
 X-rays
 Hydrostatic Pressure
(5,000 – 10,000 atm)
 UV radiation

26.  Acids and bases disrupt salt bridges held together by ionic
charges
 Organic solvents such as acetone alcohols denature proteins
by disrupting hydrophobic interactions
 Heavy metal salts usually contain Hg+2, Pb+2, Ag+1 Tl+1,
Cd+2 and other metals with high atomic weights. Since salts
are ionic they disrupt salt bridges in proteins

27.  Chaotropic agents (Urea 6 – 8 mol/l , Guanidinium chloride
6 mol/l) decrease the net hydrophobic effect of hydrophobic
regions because of a disordering of water molecules adjacent
to the protein. This solubilises the hydrophobic region in the
solution, thereby denaturing the protein.
 Detergents such as sodium dodecyl sulphate denature
proteins by associating with the non-polar groups of protein,
thus interfering with the normal hydrophobic interactions
 Changing pH denatures proteins because it changes the
charges on many of the side chains. This disrupts electrostatic
attractions and hydrogen bonds

28.  Heat : Proteins can also be denatured by heat. Heat increase
molecular motion which can disrupt the attractive forces.
 UV radiation supplies kinetic energy to protein molecules,
causing their atoms to vibrate more rapidly and disrupting
relatively weak hydrogen bonding and dispersion forces.
 Violent shaking also denatures protein. We see this clearly
in the whipping of egg whites.
 Hydrostatic Pressure denature the protein by de stabilizing
hydrophobic and electrostatic interaction