The document provides an extensive overview of protein structures, detailing the four main levels of organization: primary, secondary, tertiary, and quaternary, which are essential for the functional conformation of proteins. It explains the role of amino acids, types of chemical bonds (such as peptide bonds, hydrogen bonds, and disulfide bonds), and the importance of protein folding in determining the final shape and function of proteins. Additionally, it discusses the biochemical mechanisms involved in protein synthesis, assembly, and the relationships between structural forms like α-helixes and β-pleated sheets.

1. PROTEIN
STRUCTURE OF PROTEIN
- Primary
- Secondary
- Tertiary and
- Quaternary structures
By,
A.S. Ramya
M.Sc. Microbiology
SemII, LDC

2. AMINO ACID: Basic unit of protein

3. 20 DIFFERENT AMINO ACIDS
• Nonpolar,hydrophobic
• Polar, charged
• Polar, uncharged

4. PROTEIN
• Proteins are complex organic nitrogenous compound, which
are large biomolecules, or macromolecules.
• It is an essential part of all living organisms, especially as
structural components of body tissues such as muscle, hair,
wool etc., other proteins may be enzymes, hormones or
oxygen carriers and antibodies.
• Protein consisting of one or more long chains of amino acid
residues. Proteins are made of 20 amino acids linked by
peptide bonds.

5. • DISTRIBUTION OF AMINO ACID IN PROTEIN
• The distribution of the 20 amino acid is not uniform in all proteins.
• Eg. 40% of weight of fibroin and 25% by weight of collagen are
accounted of glycine.
• Human serum albumin with 585 amino acid residues has one
tryptophan moiety.
• LOCATION OF AMINO ACID IN PROTEIN
• Amino acid with uncharged polar side chain are relatively
hydrophilic and are usually on the outside of the protein,
while the side on nonpolar or hydrophobic amino acids tend to
cluster together on the inside.
• Amino acids with acidic or basic chains are very polar, and they
are nearly always found on the outside of the protein molecules.

6. PEPTIDE BOND
• The amino acids units are linked together through the
carboxyl and amino groups to produce primary structure of
the protein chain .The bond between two adjacent amino
acids is a special type of amine bond, known as peptide bond
and the chain thus formed is called a peptide chain.

7. • N- AND C- TERMINALS- Each amino acid in the chain is
termed a residue. The two ends of the peptide chain are
named as amino terminal ( N- terminal ) and carboxyl
terminal ( C- terminal).
• These amino acid with free amino group is called N- terminal
amino acid and one with free carboxyl group are called C-
terminal amino acid.
• The two terminal groups, one basic and another acidic are
the only ionisable groups of any peptide chain except those
present in the side chain.

8. • The four atoms (C, O, N, H)
of the peptide bond form a
rigid planar unit. There is no
freedom of rotation about the
C-N bond. On the contrary,
the 2 single bonds on either
side of the rigid peptide unit,
exhibit a high degree of
rotational freedom.

9. STEREOCHEMISTRY OF PEPTIDE CHAIN
• All proteins are made up of L-configuration. This fixes steric
arrangement at the α-carbon atom. The dimensions of the
peptide chain are known to be 7.27A˚.

10. PROTEIN CONFIGURATION
• Based on degree of complexity of the protein molecule it
has categorized into 4 basic structural levels of
organisation.
• First defined by linderstrom-lang often referred to as
primary, secondary , tertiary, quaternary structure.(1º, 2º,
3º, 4º)
• 1º, 2º, 3º structural levels can exits in molecules composed
of single polypeptide chain, whereas 4º involves
interactions of polypeptides within a multichained protein
molecule.

12. Primary structure (Amino acid sequence)
consists of one or more linear chains of
number of aminoacid units
↓
This unfold structure often assumes helical
shape to produce 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, cetain
protein has subunits (single protein
molecule) interact with each other in a
specific manner to form a protein complex.
Multimeric structure of different
monomer.

13. CHEMICAL BOND INVOLVED IN PROTEIN
STRUCTURE
• Protein synthesis is a multiple dehydration process.
• PRIMARY BOND ( peptide bond, -CO-NH- ) –
• The principal linkage found in all proteins is a covalent peptide
bond.
• Peptide bond is the backbone of the protein chain
• It is a specialised amine linkage where C atom of –COOH
group of one amino acid is linked with N atom of –NH2 group of
the adjacent amino acid releasing a molecule of water (H2O).
• This is a dehydration synthesis reaction (also known as a
condensation reaction), and usually occurs between amino
acids.

14. • SECONDARY BONDS-
• Many of the properties of proteins do not coincide with the
linear chain structure, thus indicating that a variety of bonds
other then the peptide exist in them and they holding the
chain in its natural configuration.

15. • 1. DISULFIDE BOND (-S-S-)
• In addition to peptide bond, a
second type of covalent bond
found between amino acid residues
in proteins and polypeptide in the
disulfide bond, which is formed by
the oxidation of the thiol or
sulfhydryl groups (-SH) of two
cysteine residues to yield a mole
of cystine, an amino acid with a
disulfide bridge.

16. • Disulfide Bond strength 50 kilocalories per mole and bond
length of about 2A˚ between two sulfur atoms.
• 2 cysteine residues located some distance apart in the
polypeptide chain requires the polypeptide to folded back on
itself to bring the sulfur groups close together.
• Even a slight extension breaks the sulfide bond completely.
They, therefore only stabilize the tertiary structure.
• Eg . Insulin

17. • 2. HYDROGEN BOND ( >CO….HN< ) - A hydrogen
bond is the electrostatic attraction between two polar
groups that occurs when a hydrogen (H) atom covalently
bound to a highly electronegative atom such
as nitrogen (N), oxygen (O), experiences the electrostatic
field of another highly electronegative atom nearby.
• The formation of hydrogen bond is due to the tendency of
hydrogen atom to share electrons with two neighbouring
atoms O and N.

18. • For Eg. The carbonyl oxygen of one
peptide bond shares its electrons with
the hydrogen atom of another peptide
bond.
• An interaction sets in between a C=O
group and the proton of an NH group if
these groups come within a distance of
about 2.8 A˚.
• Hydrogen bonds are relatively weak
linkages but many such bonds
collectively exerts considerable force and
help in maintaining the helical structure
or secondary structure.
• Eg. Keratin of wool, silk fibroin.

19. HYDROPHILIC HYDROPHOBIC

20. • 3. NONPOLAR OR HYDROPHOBIC BOND –
• Many aminoacids (like alanine, valine, leucine, isoleusine,
methionine, tryptophan, phenylalanine, tyrosine) have side chains
or R groups which are essentially hydrophobic.
• This R groups can unite among themselves with elimination of
water to form linkages between the chains or between different
chains. The assosiation of various R groups in this manner leads
to a relatively strong bonding.
• It also serves to bring together groups that can form hydrogen
bonds or ionic bonds in the absence of water.
• Each type of linkage helps in the formation of the other.
• The hydrophobic bond also play an important role in other
interactions. E.g. the formation of enzyme substrate complexes
and antibody antigen reactions.

21. • 4. IONIC OR ELECTROSTATIC BOND –
• Ions possessing similar charge repel each
other whereas the ions having dissimilar
charge attract each other.
• Another instance of ionic bonding may be
the interaction between the acidic and
basic groups of the constituent amino
acids.
• Thus, ionic bonds between positively
charged groups( side chains of lysine,
arginine, histidine) and negatively charged
aspartic and glutamic acids) do occur.

22. • When two oppositely charged groups are brought close
together, electrostatic interactions lead to strong attraction,
resulting in the formation of electrostatic bond.
• In fact ionised group are more frequently found stabilising
interactions between protein and other molecules.
• Although these ionic bonds are weaker than the hydrogen
bonds, are regarded as responsible for maintaining the
folded structure (or tertiary structure) of globular protein.

23. PROTEIN ASSEMBLY
• occurs at the ribosome
• involves polymerization of amino acids attached to tRNA
• yields primary structure

24. PRIMARY STRUCTURE
• The primary structure of a protein refers to the linear
sequence of amino acids in the polypeptide chain.
• The main mode of linkage of the amino acids in the
proteins is the peptide bond which links the α- carboxyl
group of one amino acid residue to the α- amino
group of the another.

25. RAMACHANDRAN PLOT
• Ramachandran Plot is a way to visualize dihedral angles ψ against
φ of amino acid residues in protein
structure. Ramachandran recognized that many combinations of
angles in a polypeptide chain are forbidden because of steric
collisions between atoms.
• TORSION ANGLE- A dihedral angle is the angle between two
intersecting planes
• PHI AND PSI ANGLES- The two torsion angles of the polypeptide
chain, also called Ramachandran angles(after the Indian physicist
who first introduced the Ramachandran plot), describe the rotations
of the polypeptide backbone around the bonds between N-Cα
(called Phi, φ) and Cα-C (called Psi, ψ).

27. • Peptide unit is a planar one. C=O and NH group of amino acid
is lie in a plane.
• With respect to the middle Cα the 2 peptide unit (C=O and NH)
are oriented to one another. Thus, this Cα carbon atom
connected with this 2 planar peptide unit.
• Phi is the tortion angle about N-Cα
• Psi is the tortion angle about Cα-C
• The N-Cα and Cα –C has a single bond so it can be twisted or
rotated. The tortional angle between two planes represent to
what extent this groups are twisted.

29. • In this graph some areas are shaded means that some phi
and psi combination is not allowed because of steric clashes
of atoms are more and energetically unfavourable.
• Shaded regions showing different types of clashes are
posssible.

31. • Amino acid has own conformation in these phi and psi
combination is represented by black spots
• White regions are favourable regions. Black spots in these
white regions assumes that this phi and psi combination
represent good configuration of amino acids (i.e) helix, strand
• Blue regions are allowed regions.
• Any phi and psi values of a given sequence fall in the
Ramachandran plot.
• In shaded regions also some black points. Some reason like
glycine have no side chain and it is highly flexible and
number of clashes less, no unfavourable region, proline have
special orientation, no unfavourable region.

33. • Less unfavoured and no clashes in the region are called
outlayers.
• Ramachandran plot validates the protein structure and
guides the structure confirment.
• Simplest and most sensitive means for accessing the
quality of the protein model.

34. PROTEIN FOLDING
• occurs in the cytosol
• involves localized spatial
interaction among primary
structure elements, i.e. the
amino acids
• Therefore the protein can fold
and orient the R groups in
favorable positions
• yields secondary structure
• Weak non-covalent
interactions will hold the
protein in its functional shape
– these are weak and will
take many to hold the shape

35. • Proteins shape is determined by the sequence of the amino
acids
• The final shape is called the conformation and has the
lowest free energy possible
• Denaturation is the process of unfolding the protein
– Can be down with heat, pH or chemical compounds
– In the chemical compound, can remove and have the
protein renature or refold

36. REFOLDING
• Molecular chaperones are small proteins that help guide the
folding and can help keep the new protein from associating
with the wrong partner.

37. SECONDARY STRUCTURE
• If the peptide bond are the only type of linkage present in the
proteins, these molecules would have behaved as irregularly
coiled peptide chain of considerable length.
• But the globular proteins have regular characteristic properties,
indicating the presence of regular coiled structure in these
molecules.
• This secondary structure is non-linear and 3 dimensional.
• Formed and stabilized by hydrogen bonding, electrostatic and
vander Waals interactions
• This involves folding of the chain which mainly due to the
presence of hydrogen bonds. Thus, folding and hydrogen bonding
between neighbouring amino acids results in the formation of a
rigid and tubular structure called a helix.

38. • Based on nature of hydrogen bonding, two main types of
secondary structure, the α-helix and the β-strand or β-pleated
sheets, were suggested in 1951 by Linus Pauling and
coworkers.
• The unstretched protein molecules formed a helix (which are
called the α-form)
• The stretching caused the helix to uncoil, forming an
extended state (which he called the β-form)
• They have a regular geometry, being constrained to specific
values of the dihedral angles ψ and φ on the Ramachandran
plot.

40. α-helix
• The α-helix is a rod like structure. Tightly
coiled polypeptide chain forms the inner part
of the rod, an the side chains extend outward
in a helical array.
• The α-helix is stabilised by hydrogen bonds
between the NH and CO groups of the peptide
chain, assumes a rod- like structure.
• A very interesting feature of the helix, besides
the periodicity, is the fact that the carbonyl
group of every peptide bond is in position to
form a hydrogen bond with the amine group of
the peptide bond in the next turn of the helix.

42. • The amino acid residue in an α-helix have conformations
with φ (phi) = -60º and ψ (psi)= -45º to -50º.
• The has a pitch of 5.4A˚ and contains 3.6 amino acids per
turn of helix , thereby giving rise per residue of 1.5A˚
• A α-helix can be right handed(clockwise) or left handed
(anticlockwise).
• Biologically functional proteins do not usually exhibit cent
percent α helix structure. Some have a high percentage of
their residues in helical structure. e.g. hemoglobin,
myoglobin; others have low percentage e.g. cytochrome C,
chymotrpsin.

43. ᵦ-pleated sheet
• ᵦ-pleated sheet in which the peptide chain is bent to form
zig-zag conformation.
• The pleated sheet structure depends on intermolecular
hydrogen bonding, although intramolecular hydrogen bonds
are also present.
• The pleated sheet structure is formed by the parallel
alignment of a number of polypeptide chain in a plane, with
hydrogen bonds between the C=O and –N-H groups of
adjacent chains.

44. • In the secondary structure, all the R groups will be facing
outwards. The β sheet structure are quite common in nature
and are favoured by the presence of aminoacids, glycine,
alanine.
• Silk and certain synthetic fibres such as nylon and orlon
are composed of β-structure
• Although the pleated sheets associated with structural
proteins, it is also known to occur in 3-dimensinal structures
of certain globular proteins, eg. The enzyme lysozyme and
carboxypeptidase A.

45. The β- pleated sheet differs markedly from the
rodlike α – helix:
• The polypeptide chain in a β- pleated sheet, called a β-strand, has
fully extended conformation, rather than being tightly coiled as in the
α- helix.
• The axial distance between adjacent amino acids in pleated sheets is
3.5 A˚, in the contrast with 1.5 A˚ for the α-helix.
• β-Sheet is stabilized by hydrogen bonds between NH and CO groups
in different polypeptide strands, where as, in α-helix, the hydrogen
bonds are between NH and CO groups in the same strand.
• In Alpha Helix -R groups of the amino acids are oriented outside of
the helix while in Beta Pleated Sheet -R groups are directed to both
inside and outside of the sheet.
• Alpha helix prefers Ala, Leu, Met, Phe, Glu, Gln, His, Lys, Arg amino
acids. Beta sheet prefers Tyr, Trp, (Phe, Met), Ile, Val, Thr, Cys.

46. BETA STRANDS CAN FORM PARALLEL OR
ANTIPARALLEL BETA-SHEETS

47. PARALLEL β PLEATED SHEET. ANTIPARALLEL β PLEATED SHEET.
Parallel – run in the same direction
with longer looping sections
between them (B)
Anti-parallel – run in an opposite
direction of its neighbor (A)
If the N terminal ends of all the
participating polypeptide chains lie on
the same edge of the sheet, with C
terminal ends on the opposite edge,
the structure is known as parallel β
pleated sheet.
In contrast, if the direction of the chains
alternates so that the alternating chains
have their N terminal ends on the same
side of the sheet, while their C terminal
ends lie on the opposite edge, the
structure is known as the antiparallel β
pleated sheet.

48. • Both parallel and anti-parallel
sheets have similar
structures, although the
repeat period is shorter
(6.5 A˚ ) for the parallel
conformation in comparison
to anti-parallel conformation
(7 A˚ )

49. Irregularities in regular structure
• A β-bulge is a common noncompetitive irregularity found in
antiparallel β-sheets.
• It occurs between two normal β structure hydrogen bonds
and involves two residues on one strand and one on other.

50. SUPER SECONDARY STRUCTURE OF PROTEINS
• There are 4 types of super
secondary structures in
proteins. They are,
• β-hairpin turn- two anti-
parallel sheets linked by
hairpin loop
• β-meander- three anti-
parallel sheets linked by
hairpin loop
• α-hairpin- two helices linked
by hairpin loop
• α-β-motif- two anti-parallel
sheets linked by a helix

51. PROTEIN PACKING
• occurs in the cytosol
• involves interaction between secondary structure elements and
solvent
• yields tertiary structure

52. TERTIARY STRUCTURE
• Tertiary structure refers to the three-dimensional structure
of monomeric and multimeric protein molecules.
• The α-helixes and β-pleated-sheets are folded into a
compact globular structure.
• It is a compact structure with hydrophobic side chains held
interior while the hydrophillic groups are on the surface of the
protein molecule.
• Tertiary structure is stabilised by hydrogen bonds, disulfide
bonds(-S-S), ionic interactions (electrostatic bonds),salt
bridge and van der Waals force.
• E.g. myoglobin, dihydrofolate reductase

54. PROTEIN INTERACTION
• Occurs in the cytosol, in close proximity to other folded and
packed proteins
• involves interaction among tertiary structure elements of
separate polymer chains

55. QUATERNARY STRUCTURE
• Quaternary structure is the assembly of two or more
polypeptide chains which are individually folded and
compacted. Multimeric structure of different monomer.
Proteins which possess quaternary structure are called as
oligomers.
• Each chain in the assembly is called as a subunit.
• Commonly occuring examples are dimers, trimers,
tetramers consisting of two, three and four polyprptide
chains, respectively. A molecule, made up of a small number
of subunits, is oligomer.

56. • The monomeric bonds are held
together by non-covalent bonds
namely hydrogen bonds,
hydrophobic interactions and
ionic bonds.
• E.g. haemoglobin made up of
four subunits, an allostreic
protein.

57. • As a result of these covalent interactions, change occur in
structure at one site of the protein molecule may cause
drastic changes in properties at a distant site. Proteins exhibit
this propery are called allosteric( protein with multiple
ligand binding site). Not all multisubunit proteins exhibit
allosteric effects, but many do.
• Importance of oligomeric proteins- these protein play a
significant role in the regulation of metabolism and cellular
function.

59. DOMAINS
• A domaindomain is a basic structural unit of a protein structure – distinct from
those that make up the conformations
• It is part of protein that can fold into a stable structure
independently
• Different domains can impart different functions to proteins
• In larger proteins each domain is connected to other domains by
short flexible regions of polypeptide.
• Proteins can have one to many domains depending on protein size.

60. PROTEINS AT WORK
• The conformation of a protein gives it a unique function
• To work proteins must interact with other molecules, usually 1 or
a few molecules
• Ligand – the molecule that a protein can bind
• Binding site – part of the protein that interacts with the ligand
– Consists of a cavity formed by a specific arrangement of
amino acids
• The binding site forms when amino acids from within the protein
come together in the folding
• The remaining sequences may play a role in regulating the
protein’s activity

61. LIGAND BINDING
• The structure of a protein, for example, may change upon
binding of its natural ligands, eg. a cofactor. In this a protein
bound to the ligand is known as holo structure, of the
unbound protein as apo structure.

62. FUNCTIONS OF PROTEIN
• The total hereditary material of the cell or genotype dictates which
type of protein the cell can produce.
• STRUCTURAL PROTEINS - are fibrous and stringy and provide
support. Eg. keratin, collagen, and elastin. Keratins strengthen
protective coverings such as skin, hair, quills, feathers, horns, and
beaks. Collagens and elastin provide support for connective
tissues such as tendons and ligaments.
• Proteins with catalytic activity( enzymes) are largely responsible
for determining the phenotype or properties of cell.
• DNA replication
• Responding to stimuli

63. • ENZYMES - are referred to as catalysts that facilitate biochemical
reactions. Eg. lactase and pepsin. Lactase breaks down the sugar
lactose found in milk. Pepsin is a digestive enzyme that works in the
stomach to break down proteins in food.
• TRANSPORT PROTEINS - are carrier proteins which move
molecules from one place to another around the body.
Eg. hemoglobin and cytochromes. Hemoglobin transports oxygen
through the blood via red blood cells. Cytochromes operate in
the electron transport chain as electron carrier proteins.
• CONTRACTILE PROTEINS - are responsible for movement.
Eg. actin and myosin. These proteins are involved
in muscle contraction and movement.

64. • HORMONAL PROTEINS - are messenger proteins which
help to coordinate certain bodily activities. Eg. insulin,
oxytocin. Insulin regulates glucose metabolism by controlling
the blood-sugar concentration. Oxytocin stimulates
contractions in females during childbirth.
• ANTIBODIES - are specialized proteins involved in
defending the body from antigens. One
way antibodies counteract antigens is by immobilizing them
so that they can be destroyed by white blood cells.
• STORAGE PROTEINS - store amino acids. Eg. casein,
ferritin. casein is a milk-based protein. Ferritin stores iron in
hemoglobin.

65. LIST OF PROTEINS (AND PROTEIN COMPLEXES)
• FIBROUS PROTEIN- 3-D structure is usually long and rod shaped
Cytoskeletal proteins- keratin , myosin
Extracellular matrix proteins- collagen, elastin
• GLOBULAR PROTEIN- Compact shape like a ball with irregular
surfaces. E.g. Enzymes are globular
Plasma proteins- serum albumin
Acute phase proteins- C-reactive protein
Hemoproteins- Hemoglobin (oxyhemoglobin and
deoxyhemoglobin)
Transmembrane transport proteins- Glucose transporter (ion
channel)

66. • Hormones and growth factors
Growth factors - Epidermal growth factor (EGF)
Fibroblast growth factor (FGF)
Peptide hormones- Insulin, oxytoxin
Steroid hormones- Estrogens, Progesterones
• Receptors
Transmembrane receptors- Rhodopsin
Intracellular receptors- Estrogen receptor

67. • DNA-binding protein- Histones, Protamines
• RNA-binding protein- SRRT (Serrate RNA effector molecule
homolog)
• Immune system proteins- Immunoglobins,
Major histocompatibility antigens, T cell receptor
• Nutrient storage/transport- Ferritin
• Chaperone proteins- GroEL %
• Enzymes

68. TOOLS
• Blastp
• CASTp
• ClustalO
• CyanoBase
• CYS-REC
• DIVEIN
• Double prediction method
• Expasy

69. • PDBsum tool
• Protscale
• Protparam
• Putative phosphorylation site prediction
• RAMPAGE
• SAVES
• SWISS-Model
• VADAR

70. Thank you