Introduction   Definition   Levinthal’s Paradox   Relation between folding & amino acid sequence   Factors affecting protein folding   Biophysical aspects -Hierarchy in protein structure -Thermodynamic consideration   Conclusion   References

1. PROTEIN FOLDING
By
KAUSHAL KUMAR SAHU
Assistant Professor (Ad Hoc)
Department of Biotechnology
Govt. Digvijay Autonomous P. G. College
Raj-Nandgaon ( C. G. )

2. CONTENTS
• Introduction
• Definition
• Levinthal’s Paradox
• Relation between folding & amino acid sequence
• Factors affecting protein folding
• Biophysical aspects
-Hierarchy in protein structure
-Thermodynamic consideration
• Conclusion
• References

3. INTRODUCTION
• Proteins are formed on the ribosome as linear polymer of
amino acids.
• This polypeptide must fold during & following synthesis to
take up its native conformation.
• This implies that a protein primary structure dictates its
three dimensional structure.
• The spatial arrangement of atoms in protein is called
conformation.

4. Figure: Different level of Protein structure

5. DEFINITION
It can be defined as the ability of a primary protein structure to
fold spontaneously to its native conformation (characteristics
& functional 3-Dimensional structure) without any information
other than the amino acid sequence.

6. LEVINTHAL’S PARADOX
• A newly synthesized polypeptide chain can theoretically assume
high number of conformation.
• Let’s assume 10100 different conformation for a polypeptide and if
each conformation were sampled in every ~10-13 seconds, it
would take about 1077 years to sample all possible conformation.
• But experimentally it takes a few seconds or up to a minute for
completion of process.
• Thus protein folding can not be a completely random & error
process. There must be short cuts.
• This was first pointed out by CYRUS LEVINTHAL in 1968,
which is also called as Levinthal’s Paradox.

7. RELATION BETWEEN FOLDING & AMINO
ACID SEQUENCE
• Each protein exist as an unfolded polypeptide or random coil when
translated from a sequence of mRNA to linear chain of amino acid.
• This polypeptide lacks any three dimensional structure.
• However amino acids interact with each other to produce a well
defined 3-dimensional structure.
• The resulting 3-dimensional structure is determined by the amino acid
sequence.
• It defines its native conformation.
• Folded proteins usually have a hydrophobic core in which side chain
packing stabilizes the folded state & charged or polar side chains
occupy the solvent exposed surface where they interact with
surrounding H2O.

8. • Formation of intra molecular H-bond provides another important
contribution to protein stability.
• The process of folding in vivo often being co- translation ally, so
that the N-terminal of the protein begins to fold while the C-
terminal portion of the protein is still being synthesized by the
ribosome.
• Protein folding may involve covalent bonding in the form of
Disulfide bond, formed between Cystines residues.
Figure: Disulfide bond

9. • Shortly before setting into more
energetically favorable native
conformation, molecules may
pass through on intermediate
‘ Molten Globule’ state.
• The essential fact of folding,
however remains that the amino
acid sequence of each protein
contains the information that
specifies both the native structure
& the pathway to attain that state.

10. FACTORS AFFECTING PROTEIN
FOLDING
• The passage of the folded state is mainly guided by
hydrophobic interaction, formation of intra molecular H-
bonds of Van der Waal forces & it is opposed by
conformational entropy.
• The chemical interactions that counteract these effect &
stabilizes the native conformation include weak (Non
covalent) interaction & disulfide bonds.
• The non covalent interactions
-Hydrogen bond
-Hydrophobic interaction
-Van der Waal interaction

11. • When folding is initiated, much of the secondary structure present
in native protein, as small single domain proteins form within a few
seconds.
• About 200 to 460 kJ/mole required to break a single covalent bond,
whereas weak interaction can be disrupted by 4 to 30 kJ/mole.
• Hydrophobic interactions are clearly important in stabilizing a
protein conformation.
• The interior of a protein is generally a densely packed core of
hydrophobic amino acid side chains.
• Presence of polar or charged group in the protein interior have
suitable partners for H-bonding or ionic interactions.

12. Figure: H- bond
• One H-bond seems to be
contribute little to the
stability but presence of
polar group without
partners in the
hydrophobic core
destabilizes the
conformation.

13. Figure to show Hydrophobic
interaction
•The interior of a protein is
generally a densely packed
core of hydrophobic amino
acid side chains.
•They have a suitable partner
for H-bonding or Ionic
interaction due to which they
form a cluster.
•This interaction are clearly
important in stabilizing
protein structure.

14. • Van der Waal interaction are
second order bond, often found
between apolar molecules &
hydrophobic side chains of
proteins.
• This interaction plays
important role in Packing
density of Protein.
Figure: High packing density of
Protein due to Van der waal
interaction

15. BIOPHYSICAL ASPECTS
• Biophysical aspects gives a deep insight into the
principles & concepts of kinetic & structural resolutions
of fast chemical & biophysical reactions of protein
folding.
• The study of fast protein folding reactions and
understanding of the folding paradox have significantly
advance due to the recent development of new
biophysical methods which allow not only kinetic
resolution in the sub millisecond time scale but also
structural resolutions.

16. (A) HIERARCHY IN PROTEIN
STRUCTURE
• In this, the protein folding process is hierarchal.
• Local secondary structure form first, certain a sequences fold
readily into α helices and β sheets.
• This is followed by longer range interaction between, α helices that
come together to form stable super secondary structure.
• The process continues until complete domains form and the entire
polypeptide is folded.
• Folding is a hierarchal process in which it folds from its primary
structure through a series and attains its final folding.
Figure: Hierarchy in structure

17. (a) PRIMARY STRUCTURE
• The primary structure of protein refers to the number &
sequence of amino acid.
• The main mode of linkage is the peptide bond which
links the α- carboxyl group of one amino acid residue to
α-amino group of other rigid and planer peptide bond.
• Linus Pauling & Robert Corey in 1930 demonstrated that
the α- carbon atom of adjacent amino acid are separated
by three covalent bonds.
• The 4 atoms of peptide group lies trans to each other.

18. Figure: primary structure of protein

19. (b) SECONDARY STRUCTURE
• The term secondary structure refers to the local conformation of
some part of the polypeptide.
• It is the regular folding pattern of the polypeptide.
• Types are α-helix & β-conformation.
α- Helix
• The simplest arrangement of the polypeptide chain with
right planer peptide bonds.
• In this the chain is tightly wound around an imaginary
axis drawn longitudinally through middle of helix.
• The R-group of amino acid residues protrude outward
from helical backbone.

21. β - Pleated Sheet
• In the β- conformation,
the backbone of
polypeptide chains can be
arranged into a zig-zag
rather than α-helix.
• The zig-zag polypeptide
chain can be arranged
side by side to form a
structure resembling a
series of pleats.
• Formation of β-
conformation depends on
intra molecular H-
bonding.
Figure : β- conformation

22. (c) SUPER SECONDARY STRUCTURE
• Super secondary structure are also called motifs & are
particularly stable arrangements of several elements of
secondary structure & the connection between them.
• DOMAINS: polypeptide with more than a few hundred amino
acid residues often folds into 2 or more stable globular units.
Figure: Super secondary structure

23. (d) TERTIARY STRUCTURE
• The over all 3-Dimensional arrangements of all proteins
is referred to as protein’s tertiary structure.
• In this the folding of its 2 structural elements taken
place together with the spatial deposition of its side
chains.
• The tertiary structure thus involves the folding of helix
of globular protein.
• Eg. Myoglobin.
Figure: Myoglobin

24. (e) QUATERNARY STRUCTURE
• It is the next level up from tertiary structure & is
the particular spatial arrangement interaction
between 2 or more polypeptide chains.
• Eg. Hemoglobin.
Figure: Hemoglobin

26. (B) THERMODYNAMIC CONSIDERATION
• Any system has maximum stability if it is in
lowest possible energy state, this principle
applies to protein also.
• The mechanism & kinetics of the folding process
& the thermodynamic stability of the native
protein depend on polypeptide – water
interactions.
• Native proteins are marginally stable.

27. FREE ENERGY FUNNEL
• Thermodynamically, the folding process can be viewed as a
kind of free energy funnel.
• The unfolded state are characterized by a high degree of
conformational entropy.
• At the top, the number of conformations is more hence the
conformational entropy is large.
• A folding proceeds, the narrowing of the funnel represents a
decrease in number of conformational species present.

28. • Small depressions along the sides of free energy funnel
represents semi stable intermediates that can briefly slow
the folding process.
• At the bottom of the funnel, an ensemble of folding
intermediates has been reduced to a single native
conformation.
• As folding progresses, the thermodynamic path down the
funnel reduced the number of states (decrease entropy)
increases the amount of protein in the native conformation
& decrease the free energy.
• Thermodynamic stability as not evenly distributed over the
structure of protein. The molecules have regions of low &
high stability.

29. • For eg. A protein
may have two
stable domains
joined by a
segment with
lower structural
stability.
• The regions of
low stability allow
a protein to alter
its conformation
between two or
more states.
Figure: Free energy funnel

30. CONCLUSION
Protein folding is a physical process by which a polypeptide
folds into its characteristic 3- Dimensional structure.
The correct 3-Dimensional structure is essential for proper
function & the biochemical property of this folded protein
responsible for proper function.
The improper folding may cause disease.

31. REFERENCES
BOOK AUTHOR
Principle of Biochemistry David L. Nelson
Michael M. Cox
Biochemistry J. L. Jain
Biochemistry Dr. U. Satyanarayan
Fundamental of
Biochemistry
Lubert Stryer
Websites:
•www.pymol.org
•www.wikipedia.com
•www.kbiotech.com