1. Proteins

2. Learning Objectives
• Understand the importance of the levels of
protein structures
• Understand the basis for the stability of protein
structures
• Understand how proteins fold into, and unfold
from, their native conformation
• Understand the methods employed to analyze
proteins

3. Biologically Active Peptides
• Aspartame
• Glutathione
• Vasopressin
• Oxytocin
• Enkephalins
• Insulin

4. dipeptide

5. tripeptide

6. nanopeptide

7. nanopeptide

8. pentapeptide

9. 2 polypeptides

10. Protein classification based on:
Composition
• Simple proteins – no other biomolecules
present
• Conjugated proteins – presence of metal
atom or small organic molecule

12. Protein classification based on:
Solubility
• Globular – water soluble; transport function,
immune protection and catalysis
• Fibrous – water insoluble; structural functions
collagen, elastin

14. Protein classification based on:
Function

15. Protein classification based on:
Function

17. Levels of protein structure
• Primary – sequence of amino acids
• Secondary – H-bonds bet. backbone C=O and
backbone NH (pleated sheet and helix)
• Tertiary – interactions of secondary structures
• Quaternary – association of polypeptide subunits

18. Levels of protein structure

19. Levels of protein structure

21. Non-polar amino acids

22. Polar, non-charged amino acids

23. Negatively-charged amino acids

24. Positively-charged amino acids

25. The primary structure reveals the amino acid
sequence of each protein/peptide.

26. Levels of protein structure

27. Secondary structures
• The polar N-H and C=O peptide units in the
interior of the protein are held by H-bonds
• Two types which are regular structures in
protein
• a-helix and b-pleated sheet

28. a-helix features
• Coil direction – left handed or right handed
• L- amino acids favor the right hand coil
• One coil has about 3.6 aa residues; there can be several coils
with 650 aa residues(1000Å)
• Average length of helix in a globular protein is 15-20Å
• H-bonds occur between 1st O of backbone C=O to 13th H atom
of backbone NH
• The presence of the ff amino acids do not favor the helix
formation: Pro, adjacent basic or acidic amino
acids, Asn, Tyr, Ser, Thr, Ile and Cys

29. Knowing the Right Hand from the Left

31. b-pleated sheet
• Two adjacent peptides
• Parallel (both NC or CN)
• Antiparallel (N to C running in opposite
directions)
• Antiparallel more common in the structure of
proteins
• Peptides with this structure are rich in alanine
and glycine (silk fiber and spider web)

35. Supersecondary structures
or structural motifs
• The clusters are held together by favorable
non covalent interactions
• Some structural motifs of folded proteins: aa
motif; bb motif antiparallel; the Greek key
(bbbb) motif; bab motif parallel

37. Structure of triose phosphate isomerase with several bab
motifs combine to form a superbarrel (a) side view (b) top
view of the protein

38. Levels of protein structure

39. Tertiary structure
• Combination of several motifs of secondary
structures into a compact arrangement
• Noncovalent forces bring about the
interactions and stability;
– H-bonds,
– electrostatic,
– hydrophobic,
– Van Der Waal’s,
– pi-pi complexation between R-side chains
– Disulfide bonds occur between Cys residues

41. Tertiary structures are quite varied

42. Charged/polar R-groups generally map to
surfaces on soluble proteins

43. Non-polar R-groups tend to be buried in the cores of
soluble proteins
Myoglobin
Blue = non-polar R-group
Red = Heme

44. Membrane proteins have adapted to
hydrophobic environments

45. • Water excluded from the hydrophobic interior
• Folding of protein occurs after translation in
the presence of molecular chaperones
• Heat shock proteins (proteins are highly
expressed when cells are exposed to increase
in temperature) – prevent aggregation of
heat-denatured polypeptides
• Misfolded proteins aggregate and deposit in
certain organs

46. The diagram shows the role of heat-
shock proteins and a chaperonin in
protein folding.
As the ribosome moves along the
molecule of messenger RNA, a chain
of amino acids is built up to form a
new protein molecule.
The chain is protected against
unwanted interactions with other
cytoplasmic molecules by heat-shock
proteins and a chaperonin molecule
until it has successfully completed its
folding.

48. PROTEIN
DENATURATION

49. Levels of protein structure

50. Quaternary structure of proteins
• Oligomeric –two or more polypeptide chains;
subunits
• Homotypic – almost identical subunits
• Heterotypic – different subunits
• Defines the arrangement and position of each
subunit in an intact protein

52. Examples of other quaternary structures
Tetramer Hexamer Filament
SSB DNA helicase Recombinase
Allows coordinated Allows coordinated DNA binding Allows complete
DNA binding and ATP hydrolysis coverage of an
extended molecule

53. How do biochemists determine the sequence
of amino acids?
• Sanger technique
• Edmann technique
• Dansyl chloride technique

54. Sanger Technique

55. Edmann Technique

56. Large Proteins should be sequenced in smaller fragments

59. Protein isolation
• Ion exchange chrom.–based on charge
• Gel filtration chrom- based on molecular size
• Affinity chrom- selective binding to a specific
molecule
• Gel electrophoresis- Based on charge and molecular
size

60. Column
Chromatography

61. Ion-exchange
Chromatography

62. Gel/ Size -
exclusion
Chromatography

63. Affinity
Chromatography

64. Gel Electrophoresis- generally used
support medium is cellulose or thin gels made up of either polyacrylamide or agarose.
Polyacrylamide is used as
support medium for low molecular weight biochemicals such as amino acid and
carbohydrates whereas agarose for large molecules like proteins
Components of the mixture have a uniform
charge, electrophoretic mobility depends primarily on
size

65. End of lecture 