1. Amino acid and Protein Chemistry
Dr.S.Chakravarty MBBS,MD

2. Learning objectives
At the end of this lecture a student should be able to :-
Classify the amino acids based on polarity, nutritional requirements, side chains and chemical side chains
Describe the pathways of Protein turnover. Explain Ubiquitin proteosome pathway
State the four levels of protein structure and explain how the sequence of amino acids leads to the final three dimensional configuration of the protein
Explain Protein misfolding and its clinical implications
Analyse the Concept of zwitter ions and its clinical applications

3. L-Form Amino Acid Structure
Carboxylic group
COO -
Amino group a
H N 3 + H
H = Glycine
R group
CH3 = Alanine

4. STEREOISOMERISM
Compounds having same structural formula , but differ in spatial configuration are known as stereoisomerism.
Penultimate Carbon

5. Mirror Images of Amino Acid
Enantiomers

7. Amino Acids Classification
Based on polarity
Based on side chain
Based on metabolic function
Based on nutritional requirement

8. Based on Side chain I II III IV V VI VII Aliphatic Sulphur Aromatic
Classes
Side chain
Polarity
Example I II
III IV V VI
VII
Aliphatic
Sulphur
Aromatic
Hydroxyl
Acidic
Basic
Imino
Apolar
Apolar Apolar
Polar
Gly, Ala, Val, Leu, and Ileu.
Cys* and Met
Phe, Tyr & Trp
Ser, Thr and (Tyr)
Asp , Asn and Glu, Gln
Lys , Arg and His
Pro
USMLE !

9. Aliphatic side chains (non-polar)

10. Imino acid ( Non-polar)

11. Glycine :
Smallest amino acid
Inhibitory neurotransmitter
Most common amino acid of collagen
Valine, Leucine and isoleucine :
Leucine – ketogenic amino acid
2. Maple syrup urine disease
Proline – imino acid , disrupts amino acid structure

12. Aromatic amino acids
Indole ring

13. Phenyl Alanine :
Essential amino acid : forms tyrosine
Def : phenyl ketonuria ( phenyl alanine hydroxylase deficiency)
Tyrosine :
Precursor of melanin
Catecholamines – dopamine, norepinephrine and epinephrine which act as neurotransmitters
Parkinson disease : def of dopamine
synthesis of thyroxine .
Tryptophan : (indole ring)
Melatonin - circadian rhytm
Serotonin – mood alterations
Source of Niacin – def causes hartnup’s disease

14. Sulphur containing amino acids

15. Cysteine :
Used to make disulfide bonds – secondary structure of proteins
Glutathione – detoxifying agent and free radical scavenger.
Pharmacology : N –acetyl cysteine
Paracetamol toxicity
Cystic fibrosis
Cyclophosphamide toxicity – treatment
Methionine :
S-adenosyl Methionine : methyl donor
Homocysteine – derived from methionine

16. Side chains with Basic groups (polar )
Guanidino group
Imidazole group

17. Lysine and arginine – due to positive charges , main components of histone proteins.
Synthesis of nitric oxide
Release of urea
Semi-essential amino acid – positive nitrogen balance
Histidine :
Histamine – major inflammatory mediator .

18. Acidic groups and their amides (polar)

19. Side chains with –OH groups

20. 21st and 22nd aminoacid
Selenocysteine
Pyrrolysine

21. Based on Nutritional requirement
Essential Amino Acids
Not synthesized in the body
Essential in diet
Non - Essential Amino Acids
Synthesized in the body
Nutritionally essential mnemonic – MATTVILPHLY OR
PVT TIM HaLL

22. Classification based on essentiality
USMLE !
Classification based on essentiality
PVT TIM HaLL
Arginine also.

23. Based on Metabolic function

24. Classification of Amino Acids by Polarity
Acidic
Neutral
Basic
Asp
Asn
Ser
Arg
POLAR
Cys *
Tyr
His
Gln
Thr
Glu
Lys
Ala
Ile
Gly
Phe
NON-
POLAR
Trp
Val
Leu
Met
Pro
Non-polar substances likely to be inside the cell membrane , polar things OUTSIDE !
Cysteine is polar because of its thiol group but free cysteine residues are
found to associate with hydrophobic regions of proteins
USMLE !

25. Zwitter ion
An amino acid acts as a zwitterion, i.e., it can be either:
Positively charged in an acid solution
Negatively charged in an alkaline solution
Neutral at the isoelectric point.

26. cation
anion

27. Formation of Peptide Bond

28. Formation of Peptide Bonds by Dehydration
Amino acids are connected head to tail
NH2
COOH 1
NH2
COOH 2
Dehydration
-H2O
Carbodiimide
NH2
C N
COOH O H 2 1

29. Amino acids are joined by peptide bonds

30. Characteristics of PEPTIDE BOND
Partial double bond(The distance is 1.32 A which is midway b/w single bond (1.49 A)and double bond(1.27A))
Rigid and Planar
The partial double bond renders the amide group planar, occurring in either the cis or trans isomers.
 In the unfolded state of proteins, the peptide groups are free to isomerize and adopt both isomers; however, in the folded state, only a single isomer is adopted at each position (with rare exceptions).

31. The trans form is preferred overwhelmingly in most peptide bonds (roughly 1000:1 ratio in trans:cis populations)
In trans form there is no freedom of rotation
The side chains are free to rotate on either side of the peptide bond.

34. Free rotation is possible about only two of the three covalent bonds of the polypeptide backbone: the -carbon (C) to the carbonyl carbon (Co) bond, and the C to nitrogen bond .
The partial double-bond character of the peptide bond that links Co to the -nitrogen requires that the carbonyl carbon, carbonyl oxygen, and -nitrogen remain coplanar, thus preventing rotation.
The angle about the C—N bond is termed the phi (ф) angle, and that about the Co—C bond the psi () angle.
For amino acids other than glycine, most combinations of phi and psi angles are disallowed because of steric hindrance (Figure 5–1). The conformations of proline are even more restricted due to the absence of free rotation of the N—C bond

35. Numbering of amino acids in Peptides and Proteins
Free alpha amino group at one end – AMINO TERMINAL( N –TERMINAL )
The amino acid contributing the alpha amino group is called as first amino acid.
Biosynthesis of protein starts from this terminal end only.
Free carboxy group on other end – CARBOXY TERMINAL(C –TERMNAL ) –> LAST AMINO ACID

36. Amino acid sequences

37. PROTEINS Greek Proteios meaning PRIMARY BUILDING BLOCKS OF THE BODY
3/4TH DRY BODY WEIGHT = PROTEINS
CONTAIN C, H , O, N as major components, while S and P are minor constituents.
N content of ordinary proteins is 16% by weight.

38. PROTEOMICS
Proteomics is the large scale study of proteins , particularly their structures , functions and interactions.

39. THE NEXT STEP AFTER GENOMICS….

40. PEPTIDES
Proteins are made by polymerisation of amino acids through peptide bonds.
2 AA joined to form a DIPEPTIDE
3 AA -> TRIPEPTIDE
4 AA-> TERTAPEPTIDE
5- 10 AA -> OLIGOPEPTIDE
POLYPEPTIDE
>50 PROTEIN
3 AA means 20 3 COMBINATIONS = 8000 possible types of peptides.
An ordinary protein with 100 amino acid = possible combinations !!!

41. BIOLOGICALLY IMPORTANT PEPTIDES
Serial No.
Name
No of AMINO ACIDS
FUNCTION
< 10 AMINO ACIDS 1
THYROTROPIN RELEASING HORMONE( TRH )
3( GLU –HIS – PRO)
STIMULATES PITUITARY TO RELEASE TSH 2
GLUTATHIONE
3 ( γ-GLU-CYS –GLY)
REDUCED GLUTATHIONE (GSH )IS AN ANTIOXIDANT 3
OXYTOCIN 9
UTERINE CONTRACTIONS
SECRETION OF MILK 4
ADH
ANTI-DIURETIC HORMONE 5
ANGIOTENSIN I 10
RENIN –ANGIOTENSIN SYSTEM : REGULATION OF WATER AND ELECTROLYTE BALANCE 6
ANGIOTENSIN II 8 DO

42. Biology/Chemistry of Protein Structure
Primary
Secondary
Tertiary
Quaternary
Assembly
Folding
Packing
Interaction
S T R U C T U R E
P R O C E S S

43. PRIMARY STRUCTURE OF PROTEINS
The sequence of the amino acids in a polypeptide chain denotes its primary structure.
linear
ordered
1 dimensional
sequence of amino acid polymer
by convention, written from amino end to carboxyl end
a perfectly linear amino acid polymer is neither functional nor energetically favorable  folding!

44. primary structure of human insulin
CHAIN 1: GIVEQ CCTSI CSLYQ LENYC N
CHAIN 2: FVNQH LCGSH LVEAL YLVCG ERGFF YTPKT

45. Peptides Are Purified by Reversed-Phase High-Pressure Chromatography
Proteins & Peptides Must Be Purified Prior to Analysis. This is achieved by various methods like :-
Column Chromatography
Partition Chromatography
Size exclusion chromatography
Absorption Chromatography
Ion-Exchange Chromatography
Affinity Chromatography
Peptides Are Purified by Reversed-Phase High-Pressure Chromatography
Protein Purity Is Assessed by iso electric focussing and Polyacrylamide Gel Electrophoresis (PAGE)

46. PROTEOMIC TOOLS - 2D GEL ELECTROPHORESIS
1st DIMENSION – SEPARATION BASED ON CHARGE - ISO ELECTRIC FOCUSING
2nd DIMENSION – SEPARATION BASED ON MOLECULAR MASS – PAGE

47. IEF pH MW
Protein mixture P
AGE
Visualization of approx 1000 to 3000 proteins per gel COMPARE WITH PREVIOUS RECORDS

48. Human Proteome Organization(HUPO)

49. Secondary structure
The term secondary structure denotes the configurational relationships between residues which are about 3-4 amino acids apart in linear structure.
It is stabilized by non-covalent bonding

50. Secondary Structure non-linear 3 dimensional
localized to regions of an amino acid chain
formed and stabilized by hydrogen bonding, electrostatic and van der Waals interactions

51. The α-helix (Pauley and Corey 1951) Spiral structure
Polypeptides form the back bone and the side chains of amino acids extend b/w NH & C=O groups
Distance b/w adjacent a.a is 1.5 A
3.6 aa per turn
RIGHT HANDED
IT IS THE MOST COMMON AND STABLE CONFORMATION OF S POLYPEPTIDE CHAIN
Abundant in hemoglobin and myoglobin and and absent in chymotrypsin. Proline and hydroxy proline will not allow the formation of alpha –helix.

52. The spirals look like they are going down clockwise.

53. Secondary structure a -helix (Pauling and Cory 1951)

54. The beta pleated sheet
the polypeptide in beta-pleated sheet is almost fully extended.
The distance b/w adjacent a.a. is 3.5 A.
It is stabilized by hydrogen bonds between NH and C=O groups of neighbouring polypeptide segments.
Adjacent strands can run in same direction(parallel) or opposite direction(anti-parallel) with regard to amino and carboxy terminal.
Abundant in Silk (anti-parallel), Flavodoxin (parallel), Carbonic –anhydrase (both).
Beta bends may be formed in many proteins by the abrupt U-turn folding of the chain. Interchain disulfide likkages stabilize these bends.

55. Secondary structure beta pleated sheet

56. Collagen triple helix

57. Protein Folding Occurs in the cytosol
Involves localized spatial interaction among primary structure elements, i.e. the amino acids
May or may not involve chaperone proteins
ADVANTAGES :-
Tumbles towards conformations that reduce E (this process is thermo-dynamically favorable)
Yields higher structures of proteins.

58. Active sites open up!!!

59. Tertiary Structure Non-linear 3 DIMENSIONAL STRUCTURE OF A PROTEIN
Global but restricted to the amino acid polymer
Formed and stabilized by hydrogen bonding, non-covalent interactions like hydrophobic packing toward core and hydrophilic exposure to solvents.
A globular amino acid polymer folded and compacted is somewhat functional (catalytic) and energetically favorable  interaction!

62. It represents the three 3D structure of the protein.
It defines the steric relationships of a.a which are far apart from each other in the linear sequence , but are opposed each other by non-covalent interactions.
Thermodynamically , the most stable structure .
DOMAIN :- It is a term used to denote a compact globular functional unit of a protein.A domain is a relatively independent region of the protein , and may represent a functional unit.

63. Quaternary Structure of a Protein
Certain polypeptides aggregate to form one functional unit. This is called as Quaternary Structure .
non-linear
3 dimensional
global, and across distinct amino acid polymers
formed by hydrogen bonding, covalent bonding, hydrophobic packing and hydrophilic exposure
favorable, functional structures occur frequently and have been categorized .

64. Structure of Hemoglobin

65. Each polypeptide chain is termed as a subunit or monomer.
Stabilized by H-bonding, electrostatic bonds , hydrophobic interactions and Van Der Walls forces.
Each polypeptide chain is termed as a subunit or monomer.
Eg. Hemoglobin :- 2 α, 2 β chains
Creatine Kinase is a dimer
LDH is a tetramer
Aaspartate transcarbamoylase has 6 subunits.

66. STRUCTURE FUNCTION RELATIONSHIP
ENZYMES :-
Enzyme catalysis needs precise binding of the substrate to the active site of the enzymes.
This depends on structural conformation of the active sides so that it is precisely oriented for substrate binding.

67. STRUCTURE FUNCTION RELATIONSHIP
TRANSPORT PROTEINS
Hb has a quaternary str with 2 α and 2β subunits.Binding of O2 to one heme subunit facilitates oxygen binding by other subunits.
Binding of H+ and CO2 promotes release of O2 from hemoglobin. This is called as BOHR effect.
In Primary structure any amino acid change /deletion/ replacement of any one acid by another can cause abnormalities in its function.
Eg. Sickle cell anaemia :- In 2nd position of β-chain of Hemoglobin if there is Valine instead of Glutamic acid .

68. Classification of proteins
Classification based on Functions :-
1. Catalytic proteins e.g.enzymes
2. Structural proteins e.g. collagen , elastin, keratin
3. Contractile proteins e.g. myosin, actin, flagellar proteins
4. Transport proteins , e.g. haemoglobin, albumin
5. Regulatory proteins e.g. ACTH , insulin , GH
6.Genetic proteins , e.g. Histones
7. Protective proteins e.g. Immunoglobulins

69. Classification based on composition and solubility
Proteins may be divided into three major groups ; simple , conjugated and derived
i) Simple proteins –those proteins which on hydrolysis yield only AA or their derivatives.
1) Albumins:- Water soluble and coagulated by heat
e.g. egg albumin, lactalbumin
2)Globulins :- insoluble in pure water but soluble in dilute salt solutions , coagulated by heat e.g. immunoglobulins
3)Protamines :- These are water soluble, soluble in dilute acids and alkalies.Not coagulated by heat.Large no of arginine and lysine residues – strongly basic. e.g. Protamine zinc insulate

70. 4)Prolamines :- Soluble in 70-80%alcohol, insoluble in pure water
4)Prolamines :- Soluble in 70-80%alcohol, insoluble in pure water.Rich in proline but lack lysine. e.g. Zein (corn), Gliadin(wheat), Hordein(barley)
5)Lectins :- Proteins having high affinity to sugar groups e.g Lectin from Dolichos biflorus agglutinates human blood group A1 RBCs.
6)Scleroproteins:- insoluble in water and organic acids , soluble in strong acids .e.g.. Collagen of bone, keratin of hair, horn , nail etc

71. Conjugated proteins :-They are combinations of protein with a non-protein part called prosthetic group.
1.Glycoproteins :- These are proteins combined with carbohydrates. When carbohydrate content is more than 10% of the molecule , the viscosity is increased and these are called as mucoproteins. e.g. blood group antigens
2.Lipoproteins :- These are proteins attached loosely wit lipid components.eg serum lipoproteins
3.Nucleoproteins :- These are proteins attached to nucleic acids, e.g. Histones

72. 4.Chromoproteins :- These are proteins with coloured prosthetic groups.e.g. Hemoglobin (Heme – red), Flavoproteins (Riboflavin-yellow), Visual purple- Vitamin A- purple )etc
5.Phosphoproteins :-They contain phosphorus .e.g. Caesin of milk and vitellin of egg yolk
6.Metalloproteins :- They contain proteins e.g. Haemoglobin and cytochromes(Iron), Tyrosinase (copper), Carbonic anhydrase (Zinc)

73. iii)Derived Proteins :- They are degradation products of native proteins .
Progressive hydrolysis sequence –>
Proteins peptones  peptides  amino acids

74. Classification based on Nutritive Value
1)Nutritionally rich proteins or Complete proteins or First class proteins: -
They contain all essential a.a. in required proportion for ideal body growth and development.
E.g egg albumin , milk casein

75. 2. Incomplete proteins :- They lack one essential amino acid
2.Incomplete proteins :- They lack one essential amino acid. They cannot sustain body growth in young individuals even if they are able to maintain growth in adults. Eg Pulses are deficient in methionine and cereals are deficient in lysine .If both are combined in diet , good growth can be obtained.
3.Poor proteins :-They lack in many essential a.a. and a diet based on them cannot sustain the original body weight e.g. Zein of corn lacks Trp and Lys.

76. . Classification based on shape
1.Globular proteins :-They are spherical or oval in shape.(l/b <10) eg albumin, globulin
2.Fibrous proteins :- They are elongated or needle shaped and resist digestion .e.g. collagen, elastin, keratin etc.

77. Nitrogen balance = Nitrogen ingested - Nitrogen excreted
(primarily as protein) (primarily as urea)
Nitrogen balance = 0 (nitrogen equilibrium)
protein synthesis = protein degradation
Positive nitrogen balance
protein synthesis > protein degradation
Negative nitrogen balance
protein synthesis < protein degradation

78. Negative nitrogen balance
Starvation
PEM
Uncontrolled Diabetes Mellites
Def of any essential aminoacid
Infection
Surgery, Burns

79. MCQ1)A mixture of ala,arg,his,gly,glu is subjected to electrophoresis at pH 7.
-ve
Identify this amino
A.Glycine
B. Arginine
C.Glutamate
D.Valine
E.ALANINE
+ve

80. MCQ2
Several complexes in the mitochondrial ETC contain non-heme iron tightly bound to a thiol group of which amino acid?
A. Glutamine
B.Methionine
C.Tyrosine
D.Cysteine
E.Serine

81. Thank you