This document provides an overview of protein structure and folding. It discusses the four levels of protein structure - primary, secondary, tertiary, and quaternary. Common secondary structures like alpha helices and beta sheets are described. The document also introduces concepts like the Ramachandran plot, which maps allowed phi and psi dihedral angles in protein backbones. Protein folding and factors involved like molecular chaperones are also summarized. Disorders resulting from changes in protein conformation are briefly mentioned.

1. Structure Of Proteins
& Protein Folding
Problems
By_ Saurav K. Rawat
M.Sc. Chem.
(Physical
special)

2. Saurav K. Rawat
Department of Chemistry,
St. John’s College, Agra ;
Sat.
Dec.14th,2013
Presentation By_

3.  What the Proteins Are?
 Importance and Biological Functions
 Classification
 Molecular Masses of Some Proteins
 Amino Acids as Monomers of Proteins
 20 Types of Amino Acids
 4 levels of Protein Structure- viz. Primary,
Secondary, Tertiary, and Quaternary
Structures
 Corey -Pauling Rules
Structure of Peptide Bond

4.  Ramachandran Plot
 α- and β- Pleated Sheet Structures
 Stability and Folding of Protein
 Anfinsen’s Experiment, Levinthal Paradox and
Kinetics
 Hsp and Molecular Chaperons in Protein
Folding
 Probes for Conformational Detection
 Do You Know?
 Disorders Due to Conformational Change
 Quick and Hot Review
 References
 University Questions

5.  Proteins (Gr. Protiose : first of foremost)
 Berzelius (1837) and Mulder (1838) coined the term
protein.
 Proteins are macronutrients that support the growth
and maintenance of body tissues.
 Chemical composition- C-51%, O- 25%, H- 7%, S-
0.4%,
sometimes P- also present in traces.
 Amino acids are the basic building blocks of
proteins and are classified as essential or non-essential.
 Essential amino acids are obtained from protein-rich
foods such as meat, legumes and poultry, while non-essential
ones are synthesized naturally in your
body.
 According to the Centers for Disease Control and
Prevention, you should obtain 10 percent to 25

6. Importance of Proteins and
Their Biological Functions
Type Examples Occurrence/function
Contractile
Proteins
• Actin
• Myosin
• Dynein
•Thin filaments in myofibril
•Thick filaments in myofibril
•Cilia and flagella
Enzymes
• Hexokinase
• Lactatae dehydrogenase
• Cytoochrome c
• DNA Polymerase
•Phosphorylates glucose
•Dehydrogenates lactate
•Transfer electrons
•Replicates and repairs DNA
Hormones
• Insulin
•Adrenocorticotrophic
hormone
• Growth hormone
•Regulates glucose metabolism
•Regulates corticosteroid synthesis
•Stimulate growth of bones

7. Type Examples Occurrence/functio
n
Receptors
•Ion channel receptors
•G protein linked receptors
•Tyrosine kinase receptors
•Present on cell membrane and
cytoplasm and receives the
stimulations from the outer
environment so as to cell may
respond according to them.
Toxins
• Clostridium bolulinum
toxin
• Diphtheria toxin
• Snake venom
• Ricin
• Gossypin
•Causes bacterial food poisoning
•Bacterial toxin
•Enzymes that hydrolyze
phosphoglycerides
•Toxic protein of castor bean
•Toxic protein of cottonseed
Storage
proteins
• Ovalbumin
• Casein
• Ferritin
• Gliadin
• Zein
•Egg-white protein
•A milk protein
•Iron storage in spleen
•Seed protein of wheat
•Seed protein of corn

8. Type Examples Occurrence/functio
n
Defensive
proteins
•Antibodies
•Fibrinogen
•Thrombin
•Form complexes with foreign
proteins
•Precursor of fibrin in blood
clotting
•Component of clotting
mechanism
Transport
proteins
• Hemoglobin
• Hemocyanin
• Myoglobin
• Serum albumin
• Ceruloplasmin
•Transports O2 in blood of
vertebrates
•Transports O2 in blood of some
invertebrates
•Transports O2 in muscle cell
•Transports fatty acids in blood
•Transports copper in blood
Structural
•Viral coat protein
• Glycoprotein
•α- keratin
• Sclerotin
• Fibroin
•Sheath around nucleic acid
•Cell coats and walls
•Skin, feathers, nails, hoofs etc
•Exoskeletons of insects
•Silk of cocoons, spider webs

9. Classification of Proteins
Based on Conformation Based on Composition
Fibrous
Insoluble in
H2O
Globular
Soluble in H2O
•α-Keratin
•β-Keratin
•Collagen
•Myoglobin
•Hemoglobin
•Lysozyme
•Ribonuclease
•Chymotrypsin
•Cytochrome-c
•Lactate
dehydrogenase
•subtilisin
Simple Conjugated Derived
•Albumin
•Globulin
•Glutalins
•Prolamins
•Protamines
•Histones
•Scleroprotei
ns
•Nucleoprotein
•Lipoprotein
•Phosphoprotein
•Metalloprotein
•Glycoprotein
•Flavoprotein
•Hemoprotein
•chromoproteins
•Protiose
•Peptones
•Small
peptides
•Fibrin
•Metaprotei
ns
•Coagulated
proteins
Based on Nature of
AcidiMc olecules Basic
•Blood proteins •Histones

10. Molecular Mass of Some
Proteins
Protein Relative molecular mass
Insulin 5,700
Hemoglobin 64,500
Myoglobin 16,900
Hexokinase 102,000
Glycogen phosphorylase 370,000
Glutamine synthetase 592,000

12. Proteins are Linear Polymers of
Amino Acids
R1
NH3
＋ C CO
H
R2
NH C CO
H
R3
NH C CO
H
R2
NH3
＋ C COOー
H
＋
R1
NH3
＋ C COOー
H
＋
H H2O 2O
Peptide
bond
Peptide
bond
The amino acid
sequence is called as
primary structure
F
A A
G N
G
S
T
S
D
K
A carboxylic acid
condenses with an amino
group with the release of a
water

13. Amino Acid: Basic Unit of Protein
Different side chains, R, determin the
properties of 20 amino acids.
R
NH + C
COO- 3
Amino group Carboxylic
H
acid group

14. Facts About Amino Acids
 Though approximately 300 amino acids occur in
nature but only 20 make the composition of
proteins.
 All amino acids, apart from the simplest one
(glycine) show optical isomerism.
 This can result in two different arrangements viz.
D- amino acid and L- amino acid.
 With a few minor exceptions, e.g., bacterial cell wall
contains D- amino acids only the L- forms are found in
living organisms.
 Gamma Amino Butyric Acid (GABA), Histamine
serotonin, Ornithine, Citruline and β- alanine are the
amino acids, which are not found in proteins.

15. 20 Types of Amino Acids
Glycine (G)
Methionine (M)
Glutamine (Q) Serine (S) Threonine (T)
Asparatic acid (D) Glutamic acid (E)
Phenylalanine (F) Tryptophan (W) Asparagine (N)
Cysteine (C)
Proline (P)
Alanine (A) Valine (V) Isoleucine (I) Leucine (L)
Tyrosine (Y)
Lysine (K) Arginine (R)
Histidine (H)
White: Hydrophobic, Green: Hydrophilic, Red: Acidic, Blue: Basic

16. Hierarchical Nature of Protein
Structure
Primary structure (Amino acid sequence)
↓
Secondary structure （α-helix, β-sheet）
↓
Tertiary structure （Three-dimensional structure
formed by assembly of secondary structures）
↓
Quaternary structure （Structure formed by more
than one polypeptide chains）

17. Definitions of the Four Levels of
Structure
 Primary structure- refers to the covalent backbone of the polypeptide
chain and the sequence of its amino acid residues.
 The enzyme ribonuclease and the protein myoglobin function only in their
primary structure.
 Secondary structure- refers to a regular recurring arrangement in space of
the polypeptide chain along one dimension.
 Secondary structures are stabilized by H-bonds.
 Keratin (a fibrous protein found in skin) is composed of almost entirely of
α- helices, while Fibrion (silk protein) is almost entirely composed of β- sheets.
 Tertiary structure- refers to how the polypeptide chain is bent or folded in
three dimensions, to form the compact, tightly folded structure of globular
proteins.
 The interactions involved in folding include weak ionic bonds, H-bonds,
hydrophobic interactions and strong disulphide bonds b/w neighbouring
cysteine amino acids.
 Enzymes are functional with a tertiary structure only.
 Quaternary structure- refers to how individual polypeptide chains of a
protein having two or more chains are arranged in relation to each other.
Most larger proteins contain two or more polypeptide chains b/w which
linkage.

18. Primary Structure of Protein

19. • It is a globular
protein
• It contains two
polypeptide
chains
• Alpha unit has
21 amino acid
residues
• Beta subunit
has 30 amino
acid residues
• Neighbouring
cysteines are
linked by
disulphide
bond

20. Introduction to Structure of Proteins
• Unlike most organic polymers, protein molecules
adopt a specific 3-dimensional conformation in the
aqueous solution.
• This structure is able to fulfill a specific biological
function
• This structure is called the native fold
• The native fold has a large number of favorable
interactions within the protein
• There is a cost in conformational entropy of folding
the protein into one specific native fold

21. Corey- Pauling Rules
 A set of rules, formulated by Robert Corey and
Linus Pauling in 1951, that govern the secondary
nature of proteins. The Corey-Pauling rules are
concerned with the stability of structures provided
by hydrogen bonds associated with the –CO-NH–
peptide link. The Corey-Pauling rules state that:
(1) All the atoms in the peptide link lie in the same
plane.
The planarity of the link is due to delocalization of
pi electrons over the O ,C and N atoms and the
maintenance of maximum overlap of their p-orbitals.
(2) The N, H, and O atoms in a hydrogen bond are
approximately on a straight line.
(3) All the CO and NH groups are involved in
bonding.
Two important structures in which the Corey-

22. Scheme Showing Peptide
Structure

23. Structure of the Peptide Bond
 Structure of the protein is partially dictated
by the properties of the peptide bond
 The peptide bond is a resonance hybrid of
two canonical structures
 The resonance causes the peptide bonds
 be less reactive compared to e.g. esters
 be quite rigid and nearly planar
 exhibit large dipole moment in the favored trans
configuration

25. The Rigid Peptide Plane and
the Partially Free Rotations
 Rotation around the peptide bond is not permitted
 Rotation around bonds connected to the alpha
carbon is permitted
 f (phi): angle around the -carbon—amide
nitrogen bond
 y (psi): angle around the -carbon— carbonyl
carbon bond
 In a fully extended polypeptide, both y and f are
180°

27. Distribution of f and y Dihedral Angles
• Some f and y combinations are very unfavorable because
of steric crowding of backbone atoms with other atoms in
the backbone or side-chains
• Some f and y combinations are more favorable because of
chance to form favorable H-bonding interactions along the
backbone
•Ramachandran plot shows the distribution of f and y
dihedral angles that are found in a protein
•shows the common secondary structure elements
• reveals regions with unusual backbone structure

28. Ramachandran Plot

29.  Secondary structure refers to a local spatial
arrangement of the polypeptide chain
 Two regular arrangements are common:
 The  helix
 stabilized by hydrogen bonds between nearby residues
 The  sheet
 stabilized by hydrogen bonds between adjacent
segments that may not be nearby
 Irregular arrangement of the polypeptide chain is
called the random coil

30. Basic structural units of proteins:
Secondary structure
α-helix β-sheet
Secondary structures, α-helix and β-
sheet, have regular hydrogen-bonding
patterns.

31. The  helix
 The backbone is more
compact with the y dihedral
(N–C—C–N) in the range (
0 < y < -70)
 Helical backbone is held
together by hydrogen bonds
between the nearby
backbone amides
 Right-handed helix with 3.6
residues (5.4 Å) per turn
 Peptide bonds are aligned
roughly parallel with the
helical axis
 Side chains point out and are
roughly perpendicular with
the helical axis

34. The  helix: Top View
• The inner diameter of the helix
(no side-chains) is about 4 – 5
Å
• Too small for anything to fit
“inside”
• The outer diameter of the
helix (with side chains) is 10 –
12 Å
• Happens to fit well into the
major groove of dsDNA
• Residues 1 and 8 align nicely
on top of each other
• What kind of sequence
gives an helix with one
hydrophobic face?

35. Sequence Affects Helix
Stability
 Not all polypeptide
sequences adopt -helical
structures
 Small hydrophobic residues
such as Ala and Leu are
strong helix formers
 Pro acts as a helix breaker
because the rotation around
the N-Ca bond is impossible
 Gly acts as a helix breaker
because the tiny R-group
supports other conformations

36. The Helix Macro-
Dipole
 Peptide bond has a strong
dipole moment
 Carbonyl O negative
 Amide H positive
 All peptide bonds in the 
helix have a similar
orientation
 The  helix has a large
macroscopic dipole
moment
 Negatively charged
residues often occur near
the positive end of the helix
dipole

37.  Sheets
 The backbone is more extended
with the y dihedral
(N–C—C–N) in the range ( 90
< y < 180)
 The planarity of the peptide
bond and tetrahedral geometry
of the -carbon create a pleated
sheet-like structure
 Sheet-like arrangement of
backbone is held together by
hydrogen bonds between the
more distal backbone amides
 Side chains protrude from the
sheet alternating in up and
down direction

39. Parallel and Antiparallel 
Sheets
 Parallel or antiparallel orientation of two chains
within a sheet are possible
 In parallel  sheets the H-bonded strands run in
the same direction
 In antiparallel  sheets the H-bonded strands
run in opposite directions

41. Structure of -
Keratin in Hair

42. Chemistry of Curly Hair

43. Structure of Collagen
 Collagen is an important constituent of connective tissue: tendons, cartilage, bones,
cornea of the eye
 Each collagen chain is a long Gly- and Pro-rich left-handed helix
 Three collagen chains intertwine into a right-handed superhelical triple helix
 The triple helix has higher tensile strength than a steel wire of equal cross section
 Many triple-helixes assemble into a collagen fibril

44. Collagen
Fibrils

45. Silk Fibroin
 Fibroin is the main protein in silk from moths and spiders
 Antiparallel  sheet structure
 Small side chains (Ala and Gly) allow the close packing of sheets
 Structure is stabilized by
 hydrogen bonding within sheets
 London dispersion interactions between sheets

46.  Turns (Hairpins)
 -turns occur frequently whenever strands in  sheets change the
direction
 The 180° turn is accomplished over four amino acids
 The turn is stabilized by a hydrogen bond from a carbonyl oxygen to
amide proton three residues down the sequence
 Proline in position 2 or glycine in position 3 are common in -turns

47. • Tertiary structure refers to the overall spatial arrangement of atoms in a
polypeptide chain or in a protein
• One can distinguish two major classes
– fibrous proteins
¤ typically insoluble; made from a single secondary structure
– globular proteins
¤ water-soluble globular proteins
¤ lipid-soluble membraneous proteins

48. Favorable Interactions in
Proteins
• Hydrophobic effect
– Release of water molecules from the structured solvation layer
around the molecule as protein folds increases the net entropy
• Hydrogen bonds
– Interaction of N-H and C=O of the peptide bond leads to local
regular structures such as -helixes and -sheets
• London dispersion
– 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, esp. buried in the hydrophobic environment strongly
stabilize the protein

49. Motifs (folds)
Arrangements of several secondary
structure elements

50. Three-dimensional structure of
proteins
zzzzz
Tertiary structure
Quaternary structure

51. • Quaternary structure is formed by spontaneous
assembly of individual polypeptides into a larger
functional cluster together. Proteins with two or more
polypeptide chains are known as oligomeric proteins.

52. Close relationship between protein
structure and its function
Example of enzyme reaction Hormone receptor Antibody
substrates
enzyme A
B
A
enzyme
Binding to A
Digestion
of A!
Matching
the shape
to A
enzyme

54. Protein Stability and Folding
•A protein’s function depends on its three-dimensional structure.
•Loss of structural integrity with accompanying loss of activity is called
denaturation
•Proteins can be denatured by
• heat or cold; pH extremes; organic solvents
• chaotropic agents: urea and guanidinium hydrochloride

55. Ribonuclease
Refolding/Anfinsen’s
Experiment
• Ribonuclease is a small protein that
contains 8 cysteins linked via four
disulfide bonds
• Urea in the presence of 2-
mercaptoethanol fully denatures
ribonuclease
• When urea and 2-mercaptoethanol
are removed, the protein
spontaneously refolds, and the
correct disulfide bonds are reformed
• The sequence alone determines the
native conformation
• Quite “simple” experiment, but so
important it earned Chris Anfinsen
the 1972 Chemistry Nobel Prize

56. How Can
Proteins Fold
So Fast?
 Proteins fold to the lowest-energy fold in the
microsecond to second time scales. How
can they find the right fold so fast?
 Protein folding is a very finely
tuned process. Hydrogen bonding
between different atoms provides
the force required. Hydrophobic
interactions between hydrophobic
amino acids pack the hydrophobic
residues.
 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

57.  The Levinthal Paradox and Kinetics
 Levinthal's paradox is a thought experiment, also
constituting a self-reference in the theory of protein folding.
In 1969, Cyrus Levinthal noted that, because of the very
large number of degrees of freedom in an unfolded
polypeptide chain, the molecule has an astronomical
number of possible conformations. An estimate of 3300 or
10143 was made in one of his papers.
 The Levinthal paradox observes that if a protein were
folded by sequentially sampling of all possible
conformations, it would take an astronomical amount of
time to do so, even if the conformations were sampled at a
rapid rate
(on the nanosecond or picosecond scale). Based upon the
observation that proteins fold much faster than this,
Levinthal then proposed that a random conformational
search does not occur, and the protein must, therefore, fold

58.  If we assume that a protein molecule has n amino acid residues,
that each residue has 2 bonds capable of rotation, and that
there are 3 possible conformations (ϕ or ψ angles) for each
rotatable bond in he backbone, the maximum number of possible
conformations is 32n , which is approximately equal to 10n . Since each
single bond can rotate completely in about 10-13 s, the total time
required for every formal single bond in the backbone to rotate once is
about 2×10-13s. Therefore the time required for a peptide chain to try
out every possible conformation it can assume that t=10n (2n×10-13) .
For a polypeptide chain of 6 residues t is in the range of microseconds,
for a chain of 11 residues, about 0.2s, but for a chain of 100 residues it
would be about 2×10 89s. or longer than the age of the earth. Yet
staphylococcal nuclease, which has 149 residues, requires at most 0.1
to 0.2 s. How…? Why the chain fold so quickly into native
conformation?
 Why it is not trying out all its possible conformations?
 This question is a major problem in biochemistry and researches
are going on..
 This is only a hypothesis that it works on The Principle of
cooperativety- once a weak bonds (hydrogen bonds or hydrophobic
interactions) have correctly formed in a part of polypeptide chain, they
greatly increase the probability of the formation of further correct
bonds without requiring the chain to try out all possible conformations.

59.  Heat shocked proteins (Hsp) – These proteins
are being synthesize vigorously when the cell is
on the heat, or the environment where they have
high heat.
 High heat can trigger the translation of more and
more Hsp.
 Hsp help to fold protein properly.
 There are two major classes of Hsp viz.
 Hsp 70- also called Chaparones (DnaJ-DnaK)
 Hsp 60- also called Chaparonins (GroEL- GroES)

60. Chaperones Assisted Protein
folding

61. Chaperones Prevent
Misfolding

62. Chaperonins Facilitate
Folding

63. Probes of Protein
Conformation
X-Ray Analysis
ORD- optical rotatory dispersion
CD- circular dichroism
Fluorescence
Fluorescence polarization
NMR- nuclear magnetic resonance
spectroscopy

64. Protein Structure Methods:
X-Ray Crystallography
Steps needed:
 Purify the protein
 Crystallize the protein
 Collect diffraction data
 Calculate electron density
 Fit residues into density
Pros:
 No size limits
 Well-established
Cons:
 Difficult for membrane
proteins
 Cannot see hydrogens

65. Circular Dichroism (CD)
Analysis
 CD measures the molar
absorption difference  of
left- and right- circularly
polarized light:  = L – R
 Chromophores in the chiral
environment produce
characteristic signals
 CD signals from peptide
bonds depend on the chain
conformation

66. Proton NMR spectrum of a
protein
Amides Aromatics Alphas Aliphatics Methyls

67. Structure Methods: Biomolecular
NMR
Steps needed:
 Purify the protein
 Dissolve the protein
 Collect NMR data
 Assign NMR signals
 Calculate the structure
Pros:
 No need to crystallize the protein
 Can see many hydrogens
Cons:
 Difficult for insoluble proteins
 Works best with small proteins

68. Do You Know…..?
 Collagen is the most abundant protein in
animal world and RibUlose BISphosphate
Carboxylase Oxygenase (RUBISCO) is the most
abundant protein in the whole biosphere.
 Monellin, a Protein is the sweetest chemical
obtained from an African Berry.

69. In 2003, Human genome sequence
was deciphered!
 Genome is the complete set of genes of a living thing.
 In 2003, the human genome sequencing was
completed.
 The human genome contains about 3 billion base
3 billion base pair => 6 G letters
&
1 letter => 1 byte
The whole genome can be recorded in
just 10 CD-ROMs!
pairs.
 The number of genes is estimated to be between
20,000 to 25,000.
 The difference between the genome of human and
that of chimpanzee is only 1.23%!

70. Some Common Diseases Caused by
Conformational Change in Protein
Structure
 Proteopathy (Proteo- [pref. protein]; -pathy [suff.
disease]; refers to a class of diseases in which
certain proteins become structurally abnormal, and
thereby disrupt the function of cells, tissues and organs of
the body. Often the proteins fail to fold into their normal
configuration; in this misfolded state, the proteins can
become toxic in some way (a gain of toxic function) or
they can lose their normal function.The proteopathies
(also known as proteinopathies, protein
conformational disorders, or protein misfolding
diseases), include such diseases as Alzheimer’s
disease, Parkinson's disease, Prion disease, Type 2
Diabetes, Amyloidosis,and a wide range of other

71. Sickle cell Disease- in sickle cell
hemoglobin (Hb-S) the glutamic acid
residue in the 6th position of the β- chains are
replaced by valine.
Sodium cyanate injections are given to recovery from sickle
cell anemia

72. Proteopathy Major aggregating protein
Alzheimer's disease
Amyloid β peptide (Aβ);
Tau Protein
Prion diseases (multiple) Prion protein
Parkinson's disease and
other synucleinopathies (multiple)
α-Synuclein
Familial British dementia ABri
Familial Danish dementia ADan
Type II diabetes
Islet amyloid
polypeptide (IAPP; amylin)
Cataracts Crystallins
Retinitis pigmentosa with rhodopsin
mutations
Rhodopsin

73. REFERENCES
 Harper’s Illustrated Biochemistry
 Biochemistry by Albert L. Lehninger
 Biophysical Chemistry by Gurtu & Gurtu
 Principles of Physical Chemistry by
Puri,Sharma & Pathania
 Atkins’ Physical Chemistry
 Molecular Biology by Dr. Virbala Rastogi
 Competitive Biology by K.N. Bhatia & K.
Bhatia
 Text book of biology by S. Chakrabarty
 NCERT text books of Chemistry and Biology

74. Frequently Asked University
Questions-
 Explain the structure of Protein.
 Describe the folding problems in protein.
 How protein fold?

75. The truth shall make you
free….!!!
Tribute to Deptt. Of
Chemistry

77. Thanks A Lot-
 Our HOD Sir
 Dr. Susan Ma’m,Who Gave Me This
Opportunity
 And All Respected Teachers
Special Thank Goes To-
 Dr. Girish Maheshwary Sir
 Dr. Jyoti Zack Ma’m
(Deptt. of Zoology, St. John’s
College)

78. Rawat’s Creation-rwtdgreat@
gmail.com
rwtdgreat@yahoo.co.uk
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