about protein structure

1. PROTEIN AND
PROTEIN STRUCTURE
PTD By: vijay g
(M ph pharmacology)

2.  Proteins are polymers of amino acids and made up of one
or more polypeptide chains
 Every protein in its native state has a unique three
dimensional structure which is referred to as its
conformation.
 The number and sequence of these amino acids in the
protein are different in different proteins.
 The function of a protein arises from its conformation.
 Protein structure can be classified into four levels of
organization.

5. Four levels of structural organization of proteins
Proteins are polymers of amino acids and made up of one or more
polypeptide chains.
Four levels of structural organization can be recognized in proteins:
1. Primary structure: is determined by the number and sequence of
amino acids in the protein.
2. Secondary Structures: is the conformation of polypeptide chain
formed by twisting or folding. It occurs when amino acids are linked
by hydrogen bonds to form a-helix and B-sheets.
3. Tertiary Structure: is the three dimensional arrangement of protein
structure. It is formed when alpha-helices and beta-sheets are held
together by weak interactions.
4. Quaternary structure: occurs in protein(oligomers) consisting of
more than one polypeptide chain where certain polypeptides
aggregate to form one functional protein.

6. Structural hierarchy of proteins
• Primary structure: is a linear sequence of amino acids
forming a backbone of proteins. It refers to the order in
which amino acids are linked together in the peptide chain.
e.g. Glutathione: Tripeptide : Glutamic acid-Cysteine-
Glycine
• (N-terminal end) H2N-------- COOH- (C-terminal end)
• Peptide bond :linear, planner, rigid ,partial double bond
character

7. Primary structure of proteins
Primary structure of proteins denotes the number and
sequence of amino acids in protein. The successive amino
acids are linked by peptide bond(covalent bond).
 Generally ,amino acids are arranged as a linear chain.
Each component amino acid is called a residue or moiety.
Very rarely, proteins may be a branched form or circular
form(Gramicidin). Primary structure of proteins is largely
responsible for its functions
 The branching points in the polypeptide chain may be
produced by interchain disulphide bridges (the covalent
disulphide bonds between different polypeptide chains in
the same protein) or portion of the same polypeptide chain
(intrachain). They are part of primary structure.

8.  Each polypeptide will have an amino terminal (N-terminal)
with free amino group and a carbonyl terminal ends(C-
terminal) with free carboxy group. By convention, they are
represented with amino terminal on the left and carboxy
terminal end on the right end.
 The amino acids composition of protein determines its
physical and chemical properties.
 Primary structure of proteins (sequence of amino acids) is
determined by the genes contained in DNA Primary
structure of number of proteins are known today .e.g.
Insulin, Glucagon, Ribonuclease, Growth hormones. Any
change in the Primary structure of proteins affect their
functions

11. Clinical applications of primary structure
 Clinical applications of primary structure:
1. Presence of specific amino acid at a specific
position/number is very significant for a particular
function of protein . Any change in the sequence is
abnormal and may affect the functions and properties of
proteins.
2. Many genetic diseases result from protein with
abnormal amino acid sequences. If the primary
structure of the normal and mutated proteins are known,
the this information may be used to diagnose or clinical
study of the disease.

12. Secondary Structures of proteins
• Secondary Structures :spatial arrangement of proteins by twisting of
polypeptide chain= folding /helical coiling patterns in proteins
(alpha- helix)or zig-zag linear( beta-sheet) or mixed form by
hydrogen bonding and disulphide bonds.
• Secondary Structures denotes the steric relationship of amino acids
close to each other.
• One of the form of coiling of the polypeptide chain is right handed
alpha-helix.
• Since proteins are made up of L-amino acids, the coiling of
polypeptide chain into right handed alpha-helix is facilitated.
• Super secondary Structures :indicate folding patterns in proteins
• Linus Pauling (Noble 1954)and Robert Corey (Noble 1951) proposed
alpha-helix and beta-pleated sheet structures of polypeptide chains.

13. Different kinds of secondary structures:
1. Alpha-helix (helicoid state)
2. Beta-pleated sheet (stretched state)
3. Loop regions
4. Beta-bends or beta-turns
5. Disordered regions
6. Triple helix

14. Alpha(a)-helix
• Alpha(a)-helix : is called Alpha(a) because the first
structure elucidated by
Linus Pauling (Noble 1954)and Robert Corey (Noble 1951).It
is most common spatial structure of protein.
• If a backbone of polypeptide chain is twisted by equal
amounts about each a-carbon, it forms a coil or helix .The
a-helix is a rod like structure.
• These hydrogen bonds have an essentially optimal nitrogen
to oxygen (N-O)distance of 2.8 A°. Thus,
carbonyl(CO)group of each amino acid is hydrogen bonded
to the -NH of the amino acid that is situated 4 residues
ahead in a linear sequence
• The axial distance between adjacent amino acids is 1.5 A°
and gives 3.6amino acid residues per turn of helix.

16. Characteristics of Alpha-helix:
1. The most common stable conformation formed
spontaneously with the lowest energy.
2. right or left handed Spiral/helical /tightly coiled structure
in the stable form. A right handed helix turns in the
direction that the fingers of right hand curl when its
thumb points in the direction the helix rises.
3. Stabilized by Hydrogen bonds (weak, strong enough due
to large number to stabilize alpha helix structure) of the
main chain which forms the back bone. Side chains of
amino acids extend outwards from the central axis.

17. 4. Hydrogen bonding occurs between the carboxyl oxygen of
one peptide bond and the amide nitrogen of another
peptide bond and 3 amino acid residues apart/ further
down in the chain (e.g. 5th is hydrogen bonded to 9th and
6th is bonded 10th and so on). All peptide bonds except the
first and the last in polypeptide chain participate in
hydrogen bonding.
5. Each peptide bond in the polypeptide chain participates in
intrachain hydrogen bonding.

19. 6. Each turn of Alpha-helix:
• contains 3.6 amino acids residues/turn of the helix with the
R group protruding outward radially.
• arise along the central axis of 1.5A° per residue and travels
distance of 5.4 nm/turn.
7. The right handed a-helix more stable and common than left
handed helix. Left handed helix are rare because of
presence of L-amino acid found in protein which exclude
left handedness).
8. Proline, hydroxy proline and Glycine disrupt alpha-helix
formation and introduce a kinkor a bend in the helix.
9. Large number of acidic (Asp, Glu) or basic amino acids
(Lys, Arg,His)or amino acid with bulky R group disrupt
Alpha-helix.

20. Helix destabilizing amino acids
Helix destabilizing (helix beakers)amino acids:
e.g Glycine, Proline
Proline as a helix beaker : since nitrogen of Proline
residue in a peptide linkage has no substituted hydrogen (as
it has imino NH-group instead of amino group) for the
formation of hydrogen bond with other residue, Proline fits
only the first turn of an alpha-helix. Elsewhere, it produces
bend and turn.
Glycine as a helix beaker : all bends in alpha-helix are not
caused by Proline residue but bend often occurs also at
Glycine residues as side chain of Glycine is small.

21. ❖Structural importance of alpha-helix :
Several alpha-helices can coil around one another like
a twisted twined cable forming strong stiff bundles of
fibers and give mechanical support.

22. Structural importance of alpha-helix :
• Several alpha-helices can coil around one another like a twisted
twined cable forming strong stiff bundles of fibers and give
mechanical support.
• is stabilized by the hydrogen bonds .

23. Beta-pleated sheet (Secondary structure of proteins)
When alpha-helix is stretched the hydrogen bonds are broken
and new hydrogen bonds are formed between -CO and NH-
of adjacent parallel chain / neighboring polypeptide segments
giving rise to an arrangement of the backbone of protein
molecule→ Beta-pleated sheet. (Beta-sheet appear pleated).
2. is stabilized by the hydrogen bonds .
3. Hydrogen bonding occurs between two polypeptide
chains(H-bonds are intrachain) or two regions
(neighboring segments) of a single chain of polypeptide
chain (H-bonds are interchain).
4. Composed of 2 or more segments of fully extended
polypeptide chains.

24. 5. Two polypeptide chains in a beta-pleated sheet may run in
the same direction (parallel beta-pleated sheets ) with
regard to amino and carboxy terminal ends of polypeptide
chain or in the opposite directions(anti-parallel beta-
pleated sheets).
6. Distance between adjacent amino acid residue is 3.5 A
(translation).
7. Major structural motif in fibroin of silk (anti-parallel),
flavodoxin (parallel), Carbonic anhydrase(both) ,some
regions of globular proteins like chymotrypsin,
ribonuclease.

27. The arrangement of polypeptide chains in beta-pleated
sheet conformation
❖The arrangement of polypeptide chains in beta- pleated
sheet conformation can occur two ways:
1. Parallel beta- pleated sheet
2. Anti-parallel beta- pleated sheet
➢ A beta- sheet can also be formed by either a single
polypeptide chain folding back on to itself (H-bonds are
intrachain and stabilized by intramolecular hydrogen bonding
) or separate polypeptide chains (H-bonds are interchain) .
➢ As such , the α-helix and β-sheet are commonly found in
the same protein structure . In the globular proteins , β-sheet
form the core structure.

28. Clinical application of beta-pleated sheet : Secondary
structure of proteins
❖Clinical application of beta- pleated sheet : Secondary
structure of proteins found in both fibrous and globular
proteins.
• Anti-parallel beta-sheets conformation is less common in
human proteins.
❖Occurrence of beta-sheet :
1. Silk fibroin(the best example in nature)
2. Amyloid in human tissue: a protein that accumulates in
Amyloidosis and Alzheimer’s disease .Dementia occurring
in middle age associated with this Amyloidosis .

29. Loop regions and their Importance
❖Loop regions :
• About half of the residues in a typical globular protein are
present in alpha helices or beta-sheet . Remaining residues are
present in loop or coil conformation. Loop regions though
irregularly ordered (lacking regular secondary structure) are
biologically important as they are more ordered secondary
structure.
• Loop or coiled ≠ the random coils (disordered and
biological unimportant conformation of denatured proteins).
❖Importance of loop regions : form the antigen-binding sites
of antibodies

30. Beta-bend or beta-turn and its Importance
❖Beta-bend or beta-turn or hairpin turn or reverse turn :
refers to the segment , in which a polypeptide chain abruptly
reverses direction and often connects the ends of the adjacent
antiparallel beta-strands hence they are named as beta-turn.
Globular proteins contain Beta-bends.
❖Characteristics of beta-bend(β-bend) :
1. consists of four successive amino acid residues.
2. Frequently contains Proline or Glycine or both.
3. is stabilized by Intrachain disulphide bridges and hydrogen
bonds (hydrogen bond is formed between the first amino acid
to the forth in the bend).

31. 4. occur primarily at protein surfaces and impart globular
shape (rather than linearity) to proteins.
5. Promote the formation of anti-parallel beta –pleated sheets
6. Importance of beta-bends : they help in the formation of
compact globular structure.

32. Disordered region and its Importance
Not all residues are necessarily present or ordered secondary
structure.
• Specific residues of many proteins exist in numerous
conformation in solution and thus they are called Disordered
regions.
• Many Disordered region become ordered region when a
specific ligand is bound .
• e.g. The stabilization of Disordered regions of the catalytic
sites of many enzymes when ligand is bound.
• Importance of Disordered region : gives flexibility and
performs a vital biological role.

33. Super secondary structures of proteins
❖A protein molecule may contain all types of arrangements in
different parts .
Thus , a part may form an α-helix to be followed by β-pleated
sheets which may include parallel or anti- parallel regions with
intervening β turns ,loop regions and disordered regions. Such
combinations of secondary structural features are called Super
secondary structures.
➢These grouping of certain secondary structural elements of
proteins occur in many unrelated globular proteins.

34. ❖Characteristic properties of Super secondary structures
of proteins:
1. Folding patterns involving α - helices, β -pleated sheets (which may
be parallel or anti- parallel regions with intervening β-turns) ,loop
regions and disordered regions.
2. β - α - β 2 : in this structure ,an α -helix connects two parallel
strands of β -pleated sheets. It is the most common motif.
3. β -hairpin : consists of antiparallel β -sheets joined by relatively
tight reverse turn/ short loops.
4. α-α motif: two successive anti-parallel helices packed against each
other with their axis inclined.
5. β -barrel: extended β -pleated sheets role up to form three different
types of barrels

36. 7. Globular proteins like Chymotrypsin , Myoglobin and
Ribonuclease have Super secondary structures instead of
uniform secondary structures.
8. The secondary and Super secondary structures of large
proteins are recognized as domains or motifs.

37. ❖Domains of the globular protein :
➢The term domain is used to represent the basic structural
and functional units of protein with tertiary structure(denotes a
compact globular functional unit of protein).
➢Relatively independent region and may represent a
functional unit.
➢ are usually connected with relatively flexible areas of
protein (e.g. immunoglobulin)

38. Tertiary structure
• Tertiary structure of globular proteins defines the steric
relationship of amino acids which are far apart from each
other in the linear sequence but are close in three
dimensional aspects i.e. Three-dimensional structure of
globular proteins is dependent on the primary structure.
• It is a compact structure with hydrophobic side chains held
interior while hydrophilic groups are on the surface of the
protein molecule. This arrangement ensures stability of the
molecules.
• Three-dimensional structural conformation of globular
proteins provides and maintains the functional
characteristics.

39. • Functions of globular proteins are maintained because of
their ability to recognize and interact with a variety of
molecules .
• This structure reflects the overall shape of the molecule.
• Primary structure of protein is folded to form compact,
biologically stable and active conformation i.e. a three-
dimensional globular protein. It is referred as its Tertiary
structure.
• e.g. Insulin ,Myoglobin

40. In 1953 , Frederick Sanger determined primary structure of Insulin (a
pancreatic protein hormone) and showed for the first time that a
protein has a precisely defined amino acid sequence(primary
structure). Primary structure of Human Insulin is folded to form
compact, biologically stable and active conformation i.e. a three-
dimensional globular protein. It is referred as its Tertiary structure.

41. Covalent bonds stabilizing protein structure
❖Proteins are stabilized by three types of covalent bonds:
1. Peptide bonds
2. Disulphide bonds
3. Lysinonorleucine bonds

42. ❖Lysinonorleucine bonds:
A bond formed between oxidized lysine residue with
unmodified lysine side chain to form a crosslink, both within
and between the triple helix units. e.g. Collagen

43. Non-covalent interactions in Tertiary structure of globular proteins
❖ Tertiary structure of globular proteins : refers to the three-dimensional
conformation of proteins generated and maintained by weak
bonds(valence forces) / non-covalent interactions such as
a. Hydrogen bonds : formed between -CO and NH- of two different
peptide bonds or -OH group of hydroxy amino acids(Serine etc.) and-
COOH groups of acidic amino acids Aspartic or Glutamic acid
b. Ionic bonds/electrostatic interactions/salt bridges : formed
between oppositely charged groups when they are in close vicinity . They
are also formed oppositely charged R groups of polar amino acid
residues . e.g. basic (Histidine, Arginine , Lysine)and acidic amino acids
(Aspartic acid, Glutamic acid)
c. Hydrophilic interactions: water loving groups are associated with
water
d. Hydrophobic interactions : formed between hydrophobic groups
(hydrocarbon like)of amino acids like Alanine and Phenylalanine.

44. e. Van der Waal forces : weak ,but collectively contribute maximum
towards the protein structure.

45. Hydrogen bonds
Hydrogen bonds are formed between NH- and –CO groups of peptide
bonds by sharing single hydrogen .
• Each Hydrogen bond is weak but collectively they are strong. A large
number of Hydrogen bonds significantly contribute stability to the
protein structure.

46. Hydrophobic bonds in protein structure :
➢are formed by interactions between Hydrophobic R groups(non-polar
side chains) of neutral amino acids like Alanine , Valine , Leucine,
Isoleucine, Methionine, Phenylalanine , Tryptophan by eliminating water
molecules.
➢are not true bonds.
➢ serves to hold lipophilic side chains together.
➢The occurrence of hydrophobic forces is observed in aqueous
environment wherein the molecules are forced to stay together.

47. Electrostatic bond
Electrostatic /ionic / salt bonds/salt bridges : formed between oppositely
charged groups when they are in close vicinity i.e. negatively charged
group linked to positively charged group of amino acid e.g. COO ⁻ of
Glutamic acid associates with NH₃⁺of Lysine. They are also formed
oppositely charged R groups of polar amino acid residues.

48. Van Der Waals forces
Van Der Waals forces/interactions : Electrically neutral molecules
associate by electrostatic interactions due to induce dipoles. These are
the non-covalent associations. They are very weak ,but collectively
contribute maximum towards the protein structure. They act only on
short distances. They include both an attractive and repulsive component
between both polar and non-polar side chain of amino acid residues.

50. Quaternary structure of protein
Quaternary structure of globular proteins :refers to the spatial
arrangement of subunits (polypeptide chains) linked by non-covalent
interactions in three dimensional complexes. It occurs in proteins with
two or more peptide chains
Monomer or promoter or subunits :Individual polypeptide chain of
oligomeric protein
• Monomers in Quaternary structure of globular proteins stabilized by
a. Hydrogen bonds
b. Hydrophobic interactions non-covalent bond
c. Ionic bonds /electrostatic interactions/
d. Van der Waals forces.

51. Dimer : 2 polypeptide chains (e.g. Insulin)
❖Tetramers : 4 polypeptide chains (e.g. LDH ,Hemoglobin )
❖Oligomers : proteins with 2 or more polypeptides chain

53. ❖Examples of proteins having quaternary structure :
• Hemoglobin
• Creatine kinase
• Alkaline phosphatase
Glycolytic enzymes :
a. Aldolase
b. Lactate dehydrogenase
c. Pyruvate dehydrogenase

54. Thank you