Protein folding is the physical process by which a protein chain acquires its native 3-dimensional structure, a conformation that is usually biologically functional, in an expeditious and reproducible manner.

1. PRESENTED BY- GUIDED BY-
ANISHA MUKHERJEE Mrs. SABIHA NAZ MA’AM
M.Sc SECOND SEMESTER
BIOTECHNOLOGY

2. CONTENTS
❖ What is Protein ?
❖ Structure of Proteins
❖ What is Protein Folding ?
❖ Factors affecting Protein Folding
❖ Stages of Protein Folding
➢ Primary Structure
➢ Secondary Structure
➢ Tertiary Structure
➢ Quaternary Structure
❖ Driving forces of Protein Folding
❖ Chaperones
❖ Chaperone Protein Folding Process
❖ Anfinsen Experiment
❖ Anfinsen Protein Disulphide Isomerase (PDI)
❖ How can Proteins fold so fast?
❖ The Levinthal Paradox
❖ Experimental techniques for studying Protein Folding
❖ Models of Protein Folding
➢ Framework Model
➢ Collapse Model of Protein Folding
➢ Nucleation Condensation Model
❖ Protein Folding Mechanism
❖ Protein Misfolding
➢ Diseases caused by Protein Misfolding
❖ Denaturation / Unfolding of Proteins
➢ Mechanism of Protein Unfolding
➢ How Denaturation occurs at the level of Protein Structure
❖ Reference

3. WHAT IS PROTEIN ?
Proteins are large, complex
molecules that play many critical
roles in the body. They do most of
the work in cells and are required for
the structure, function, and regulation
of the body's tissues and organs.

4. STRUCTURE OF PROTEINS
▪ Proteins have several layers of structure each of
which is important in the process of protein
folding.
▪ The first most basic level of this structure is the
sequence of amino acids themselves (primary
structure).
▪ The next layer in protein structure is the
secondary structure which includes α-Helixes
and β-sheets.
▪ The tertiary structure is the next layer in protein
structure. This takes the α-Helixes and β-sheets
and allows them to fold into a three-dimensional
structure.

5. WHAT IS PROTEIN FOLDING ?
 Proteins are amino acid chains that require their
biological & biochemical properties by folding into
unique three dimensional structures.
 The shape into which a protein naturally folds is known
as its native state, which is, in most cases determined
only by its sequence of amino acids.
 Protein folding is commonly a very fast process taking
not more than a few milliseconds to occur.
 Proteins are folded and held together by several forms of
molecular interactions . The molecular interactions
include the thermodynamic stability of the complex, the
hydrophobic interactions and the disulfide bonds formed
in the proteins.

6.  Physiological proteins exist in the “folded” or “native” state, the
state with the lowest free energy.
 Proteins unfold into a “random coil” if temperature raised or
denaturant is added (urea, GuHCl) added.

7. PROTEIN FOLDING DEPENDS ON -
❑ The solvent (water or lipid bilayer)
❑ The concentration of salt
❑ The PH
❑ The temperature
❑ The possible presence of cofactor
❑ Molecular chaperones

8. STAGES OF PROTEIN FOLDING
 The protein folding is a complex process involving four
stages, that gives rise to various 3-D protein structures
essential for diverse functions in the human body.
 The structure of a protein is hierarchically arranged, from a
primary to quaternary structure.

9. PRIMARY STRUCTURE
➢ The primary structure refers to the linear sequence of
amino acid residues in the polypeptide chain, which
determines its native conformation.
➢ Amino acids are linked together by peptide bond.
➢ 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.
➢ The amino acid composition is not as important as the
sequence.
STABILITY FACTORS :-
1) Peptide bond
2) Terminal electrostatic interaction
3) Cis/trans structure (trans isomerase)

10. SECONDARY 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.
➢ Formation of a secondary structure is the first step in the folding process that a
protein takes to assume its native structure.
➢ Characteristic of secondary structure are the structures known as alpha helices and
beta sheets that fold rapidly because they are stabilized by intramolecular hydrogen
bonds, as was first characterized by Linus Pauling.
➢ Formation of intramolecular hydrogen bonds provides another important
contribution to protein stability.
STABILITY FACTORS :-
1. Peptide bond
2.Terminal electrostatic interaction
3. H-BONDING

11. ➢ Protein secondary structure takes on the three
forms :-
Alpha helix - Formed by the hydrogen bonding of
between peptide groups within the same polypeptide chain.
 Each complete coil contains 3.6 amino acid residues.
Beta sheet - The β pleated sheet is a structure that forms
with the backbone bending over itself to form the hydrogen
bonds.
 The hydrogen bonds are between the amide hydrogen and
carbonyl oxygen of the peptide bond.
 There exists anti-parallel β pleated sheets and parallel β
pleated sheets where the stability of the hydrogen bonds is
stronger in the anti-parallel β sheet as it hydrogen bonds
with the ideal 180 degree angle compared to the slanted
hydrogen bonds formed by parallel sheets.
Turn , coil or loop

12. TERTIARY STRUCTURE
 Tertiary structure is exhibited by proteins having only one polypeptide chain.
 The alpha helices and beta pleated sheets can be amphipathic in nature, or contain a hydrophilic
portion and a hydrophobic portion.
 This property of secondary structures aids in the tertiary structure of a protein in which the
folding occurs so that the hydrophilic sides are facing the aqueous environment surrounding the
protein and the hydrophobic sides are facing the hydrophobic core of the protein.
 Secondary structure hierarchically gives way to tertiary structure formation. Once the
protein's tertiary structure is formed and stabilized by the hydrophobic interactions, there may
also be covalent bonding in the form of disulfide bridges formed between two cysteine residues.
 Tertiary structure of a protein involves a single polypeptide chain; however, additional interactions
of folded polypeptide chains give rise to quaternary structure formation.
❑ STABILITY FACTORS :-
1. Peptide bond
2. Terminal electrostatic interaction
3. H-BONDING
4. Hydrophobic interaction
5. Disulphide bond

14. QUATERNARY STRUCTURE
 Two or more polypeptide chains associate
together to produce a quaternary structure.
 It is exhibited by proteins containing more
than one polypeptide chains.
 It is formed by the combination of primary,
secondary and tertiary structures.
 Tertiary structure may give way to the
formation of quaternary structure in some
proteins, which usually involves the "assembly"
or "co assembly" of subunits that have already
folded; in other words, multiple polypeptide
chains could interact to form a fully functional
quaternary protein.
STABILITY FACTORS :-
 1. H-BONDING
 2. Ionic bond/ salt bridge

16. DRIVING FORCES OF PROTEIN FOLDING
 Folding is a spontaneous process that is mainly guided by hydrophobic
interactions, formation of intramolecular hydrogen bonds,Van derWaals
forces, and it is opposed by conformational entropy.
 The process of folding often begins co-translationally, so that the N-terminus
of the protein begins to fold while the C-terminal portion of the protein is
still being synthesized by the ribosome; however, a protein molecule may
fold spontaneously during or after biosynthesis.

17. ❑ Forces involved in Protein stabilization are :-
 Hydrogen Bonding– Interaction of N-H and C=O of the peptide bond leads to local
regular structures such as alpha-helices and beta-sheets.
 Vander Waals interactions- Medium-range weak attraction between all atoms
contributes significantly to the stability in the interior of the protein
 Electrostatic interactions– Long-range strong interactions between permanently
charged groups .
– Salt-bridges, especially buried in the hydrophobic environment strongly stabilize the protein .
 Hydrophobic Effect (the dominant force in protein folding)– Release of water
molecules from the structured solvation layer around the molecule as protein folds increases the net
entropy.
 Ionic strengths
 Disulfide bonds

18. HYDROPHOBIC EFFECT
 Protein folding must be thermodynamically favorable within a cell
in order for it to be a spontaneous reaction. Since it is known
that protein folding is a spontaneous reaction, then it must assume
a negative Gibbs free energy value.
 Minimizing the number of hydrophobic side-chains exposed to
water is an important driving force behind the folding process.
 The hydrophobic effect is the phenomenon in which the
hydrophobic chains of a protein collapse into the core of the
protein (away from the hydrophilic environment).
 The multitude of hydrophobic groups interacting within the core of
the globular folded protein contributes a significant amount to
protein stability after folding, because of the vastly accumulatedVan
derWaals forces (specifically London Dispersion forces).

19. CHAPERONES
 Chaperones are a class of proteins that aid in the correct folding of other
proteins in vivo.
 Chaperones are proteins that assist covalent folding or unfolding and
assembly or disassembly of other macromolecular structure.
 They bind to unfolded and partially folded polypeptide chains to prevent
the improper association of exposed hydrophobic segments that might lead
to non-native folding as well as polypeptide aggregation and precipitation.
 Chaperones exist in all cellular compartments and interact with the
polypeptide chain in order to allow the native three-dimensional
conformation of the protein to form; however, chaperones themselves are
not included in the final structure of the protein they are assisting in.

20. MOLECULAR CHAPERONES
 Chaperones are present when the macromolecules perform
their normal biological functions and have correctly completed
the processes of folding and/or assembly.
 Chaperones may assist in folding even when the nascent
polypeptide is being synthesized by the ribosome.
 Molecular chaperones operate by binding to stabilize an
otherwise unstable structure of a protein in its folding pathway,
 They assist the de novo folding of proteins or they form repair
machines for misfolded or even aggregated proteins, and they
are therefore especially important for the survival of cells during
stress situations.

21.  A well studied example is the bacterial
GroEL system, assists in the folding of
globular proteins.
 In eukaryotic organisms chaperones are
known as heat shock proteins .
 These are basically proteins that are involved
in the folding and unfolding of other proteins.
 Various approaches have been applied to
study the structure, dynamics and functioning
of chaperones.
 Chaperone-assisted folding is required in the
crowded intracellular environment to prevent
aggregation.
 Used to prevent misfolding and aggregation
which may occur as a consequence of
exposure to heat or other changes in the
cellular environment .

22. ❑ There are two major classes of molecular
chaperones in both prokaryotes and eukaryotes :-
❖ The Hsp70 family of 70-kD proteins.
❖ The chaperonins (large multisubunit proteins).
❑ Hsp70 PROTEINS :-
 Hsp70 proteins bind to regions of unfolded polypeptides that are rich in
hydrophobic residues, preventing inappropriate aggregation.
 These chaperones thus “protect” proteins that have been denatured by
heat and peptides that are being synthesized and are not yet folded.
 Hsp70 proteins also block the folding of certain proteins that must remain
unfolded until they have been translocated across membranes.
 Some chaperones also facilitate the quaternary assembly of oligomeric
proteins .

23. ❑ Chaperonins :-
 GroEL/GroES system in E. coli .
 Unfolded proteins are bound within
pockets in the GroEL complex.
 The pockets are capped transiently
by the GroES “lid” .
 GroEL undergoes substantial
conformational changes, coupled to
ATP hydrolysis and the binding and
release of GroES , which promote
folding of the bound polypeptide.
 Then, isomerization takes place.

24. CHAPERONE PROTEIN FOLDING PROCESS
 Unfolded substrate proteins
bind to a hydrophobic
binding patch on the interior
rim of the open cavity of
GroEL.
 Binding of substrate protein
in this manner, in addition to
binding of ATP, induces a
conformational change that
allows association of the
binary complex with a
separate lid structure,
GroES.ATP is hydrolyzed
and relase the GroES, which
promote folding of protein.

25. ANFINSEN EXPERIMENT
➢ Also known as Ribonuclease Refolding experiment .
➢Ribonuclease is a small protein that contains 8 cysteines
linked via four disulfide bonds.
➢8M Urea in the presence of 2-mercaptoethanol fully
denatures ribonuclease, leads to random coil & no activity.
➢When urea and 2-mercaptoethanol are removed, the protein
spontaneously refolds, and the correct disulfide bonds are
reformed
➢All the information necessary for folding into its native
structure is contained in the amino acid sequence of the
protein..The sequence alone determines the native
conformation.
➢Further addition of trace amounts of b-mercaptoethanol
converts the scrambled form into native form.
➢ The native form of a protein has the thermodynamically
most stable structure.

26. ANFINSEN PROTEIN DISULPHIDE ISOMERASE (PDI)
 Under optimal experimental conditions, proteins often fold more slowly in
vitro than they fold in vivo.
 Reason is that folding proteins often form disulfide bonds not present in the
native proteins, which then slowly form native disulfide bonds through the
process of disulfide interchange.
 Protein disulfide Isomerase (PDI) catalyzes this process.
 Indeed, the observation that RNase A folds so much faster in vivo than in
vitro led “Anfinsen” to discover this enzyme.

27.  Proteins fold to the lowest-energy fold in the microsecond to
second time scales. How can they find the right fold so fast?
 It is mathematically impossible for protein folding to occur by
randomly trying every conformation until the lowest-energy one
is found (Levinthal’s paradox) .
 Search for the minimum is not random because the direction
toward the native structure is thermodynamically most
favorable.
HOW CAN PROTEINS FOLD SO FAST ?

28. ❖The thermodynamics of protein folding depicted as a free-energy funnel.At
the top, the number of conformations, and hence the conformational entropy,
is large.
❖Only a small fraction of the intramolecular interactions that will exist in the
native conformation are present.
❖ As folding progresses, the thermodynamic path down the funnel reduces the
number of states present (decreases entropy), increases the amount of protein
in the native conformation, and decreases the free energy.
❖ Depressions on the sides of the funnel represent semi-stable folding
intermediates, which in some cases may slow the folding process.

29. THE LEVINTHAL PARADOX
 The Levinthal Paradox states that the number of possible
conformation available to a protein is astronomically large.
 Imagine a 100-residue protein so it has 99 peptide bonds so 198 phi and
psi angles if each of these bond angles can be one of 3 stable conformation
so maximum 3198 occure different conformations
so require long time than the age of universe to
arise at its correct native conformation .
 CONCLUSION :- folding not a random
but have specific path way.

30. ❖ EXPERIMENTAL TECHNIQUES for studying protein folding
 X-ray crystallography
 Fluorescence spectroscopy
 Circular Dichroism
❑ X Ray crystallography :-
 Crystallography is one of the more efficient and important methods for
attempting to decipher the three dimensional configuration of a folded protein.
 To be able to conduct X-ray crystallography, the protein under investigation
must be located inside a crystal lattice.
 Only by relating the electron density clouds with the amplitude of the x-rays
can this pattern be read and lead to assumptions of the phases or phase angles
involved that complicate this method.
 Steps needed :-
➢ Purify the protein
➢ Crystallize the protein
➢ Collect diffraction data
➢ Calculate electron density
➢ Fit residues into density

31. ❑ Fluorescence spectroscopy :-
 Fluorescence spectroscopy is a highly sensitive method for studying
the folding state of proteins.
 Three amino acids, phenylalanine (Phe), tyrosine (Tyr) and
tryptophan (Trp) have intrinsic fluorescence properties, but only Tyr
and Trp are used experimentally because their quantum yields are
high enough to give good fluorescence signals.

32. MODELS OF PROTEIN FOLDING
❖ FRAMEWORK MODEL –
 This suggest that folding is start with formation of secondary structure
which then interact to form a more advanced folding intermediate.
• Supported by experimental observation of rapid formation of
secondary structure during protein folding process.

33. MOLTEN GLOBULE STATE -
 The molten globule state is an intermediate
conformational state between the native and fully unfold
states of globular protein.
 Some character of molten state are :-
▪ The presence of native like content of secondary structure.
▪ The absence of specific tertiary structure produced by the tight packing of amino
acid side chain.
▪ Model for early stages of protein folding (hydrophobic collapse).

34. ❖ COLLAPSE MODEL OF PROTEIN FOLDING -
 Experimental observations indicate that protein folding begins with the formation
of local segments of secondary structure (α helices and s sheets).
 Since native proteins contain compact hydrophobic cores, it is likely that the
driving force in protein folding is what has been termed a hydrophobic collapse.
 The collapsed state is known as a molten globule, a species that has much of the
secondary structure of the native protein but little of its tertiary structure.
 During this intermediate stage, the native like elements are thought to take the form
of sub domains that are not yet properly docked to form domains.
 In the final stage of folding, the protein undergoes a series of complex motions in
which it attains its relatively rigid internal side chain packing and hydrogen
bonding while it expels the remaining water molecules from its hydrophobic core.

35. ❖ NUCLEATION CONDENSATION MODEL -
 In this model the secondary and tertiary
structure at a time made , the
hydrophobic core collapse in random
fashion and form a native structure.
 “Evidently, proteins have evolved to
have efficient folding pathways as well
as stable native conformations.”
 Nevertheless, misfolded proteins do
occur in nature, and their accumulation
is believed to be the cause of a variety
of diseases.

37. PROTEIN FOLDING MECHANISM
▪ Nascent protein non-functional
linear
▪ Native functional Nonlinear 3D
▪ Protein folding is either by; co
translational process( N-terminus
is folded while the C-terminus is
synthesizing) or after translation.

38. PROTEIN MISFOLDING
 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 protein typically contains β-sheets that are organized in a
supramolecular arrangement known as a cross β-structure.These
β-sheet-rich assemblies are very stable, very insoluble and generally
resistant to proteolysis.
 The misfolding of proteins can trigger the further misfolding and
accumulation of other proteins into aggregates or oligomers.
 The increased levels of aggregated proteins in the cell leads to
formation of amyloid-like structures which can cause degenerative
disorders and cell death.

39. DISEASES CAUSED BY PROTEIN MISFOLDING
❑ Alzheimer’s disease- Neurodegenerative condition caused by protein
misfolding.This disease is characterized by dense plaques in the brain caused
by misfolding of the secondary β-sheets of the fibrillar β-amyloid proteins
present in brain matter.
❑ Parkinson’s disease- Neurodegenerative condition caused by protein
misfolding. It mainly affects the motor system.
❑ Huntington's disease- Neurodegenerative condition caused by protein
misfolding. It is a rare, inherited disease & has a broad impact on a person’s
functional abilities and usually results in movement, cognitive (thinking) and
psychiatric disorders.
❑ Cystic fibrosis (CF)- It is a fatal disease caused by misfolding of the
cystic fibrosis transmembrane conductance regulator (CFTR) protein.
❑ Gaucher’s disease- It is caused by mutations of the GBA1 gene, which
encodes the lysosomal anchored glucocerebrosidase (GCase). GBA1
mutations commonly result in protein misfolding, abnormal chaperon
recognition & premature degradation.

40. DENATURATION / UNFOLDING OF PROTEINS
 Denaturation is a process in which a protein loses its native shape
due to the disruption of weak chemical bonds and interaction,
thereby becoming biologically inactive.
❖ Factors causing denaturation of proteins :-
 Changing pH denatures proteins.
 Certain reagents such as urea and guanidine hydrochloride
denature proteins .
 Detergents such as sodium dodecyl sulphate denature
proteins by associating with non-polar group of proteins.

41. ➢ When protein is denatured it
loses its function.
Examples :-
 A denatured enzyme ceases/stops
its function.
 A denatured antibody do not binds
to its antigen.
 The denatured state of protein does not
necessarily mean that complete
unfolding or denaturation of protein.
 Under some of conditions these proteins
exhibit both properties denaturation and
renaturation.

42. MECHANISM OF PROTEIN UNFOLDING
➢Unfolding of native proteins occur at both temperatures
higher temperature and lower temperature.
❖ Types of denaturation :-
 Heat denaturation/thermal denaturation.
 Cold denaturation.

43. HOW DENATURATION OCCURS AT THE LEVEL
OF PROTEIN STRUCTURE
 Denaturation occurs at the secondary ,tertiary and quaternary
structure but not at the primary structure level.
 When the shape is compromised and the molecule can no
longer function in its desired capacity.

44. REFERENCES
❖ Nelson and Cox – Principles of Biochemistry, 5th Edition (2009)
❖ Albert L. Lehninger – Biochemistry, Second Edition (2005).
❖ www.slideshare.net
❖ www.wikipedia.org