1. Proteins

2. Amino acids-proteins
• I. Overview
• Most diverse and abundant molecules in living
systems
• Functional components: enzymes, hormones, cell-
surface receptors
• Structural components: cell membranes, organelles;
bone, skin, muscle, connective tissue
• Other specialized roles: immunoglobulins,
hemoglobin, albumin

4. II. Structure of Amino Acids
• More than 300 amino acids known, but only 20 coded
for by DNA
• At pH 7.4 (physiological pH), amino acids exist in
zwitterionic form (positive NH3+ and negative COO-
charges).
• Classified based on side chain (R) group:
Nonpolar, Polar, Charged (acidic or basic)

7. A. Amino acids with non-polar side chains
• do not bind nor give protons
• do not form hydrogen bonds
• have hydrophobic interactions
• 1. Location of non-polar (hydrophobic) amino acids in
proteins
– In soluble proteins (aqueous environment), found in
the interior of proteins (shielded from environment)
– In membranes or other hydrophobic environments,
found on protein surface.
– Proline: side chain forms an imino group

10. B. Amino acids with uncharged polar side chains

11. • 0 charge at neutral pH
• Cys & Tyr can lose a proton at alkaline pH
• Ser, Thr & Tyr – polar –OH can form hydrogen
bonds
• Asn & Gln contain –COOH (carboxy) and –
CONH2 (carboxyamine) groups – can form
hydrogen bonds.

13. • 1. Disulfide bond:
• Side chain of Cys contains –SH group –
important active site of enzymes
• Proteins with 2 –SH groups can form a
disulphide bridge or cystine dimer (-S-S- ,
intermolecular or intramolecular).

15. B. Amino acids with uncharged polar side chains
• 2. Side chains as sites of attachments for other
compounds:
• Ser, Thr & Tyr contain polar –OH group – site of
attachment for PO4- group, for e.g. Ser side-
chain important active site component in many
enzymes
– -CONH2 group of Asn and –OH group of Ser &
Thr serve as site of attachment of
oligosaccharide chains in glycoproteins

17. C. Amino acids with acidic side chains
• Asp & Glu are proton donors.
• At neutral pH (physiological), side chains fully
ionized or dissociated (COO-) and carry a net
negative charge.
• Contribute a negative charge to proteins .
• Aspartate (aspartic acid) and glutamate
(glutamic acid).
• R groups typically have a pK< 7

21. D. Amino acids with basic side chains
• Side chains of basic amino acids accept protons
• At physiologic pH, side chains of Lys and Arg are fully ionized –
positively charged ( NH3+)
• Contribute a positive charge to proteins that contain them
• Have a pK value>7
( histones have an abundance of Arg and lys, net +ve charge)
• His -- weakly basic and partially positively charged at
physiologic pH- good buffering capacity
• In proteins, can be +ve or –ve depending on environment of
protein (important role in proteins like myoglobin).

23. E. Abbreviations and symbols for commonly occurring amino acids
3-letter abbreviation and one-letter symbol
1. Unique first letter
Cys C
Cysteine
Histidine His H
Isoleucine Ile I
Methionine Met M
Serine Ser S
Valine Val V

24. 2. Most commonly occurring amino acids have priority
Ala A
Alanine
Glycine Gly G
Leucine Leu L
Proline Pro P
Threonine Thr T

25. 3. Similar sounding names
Arg R (“aRginine)
Arginine
Asparagine Asn N (contains N)
Aspartate Asp D (“asparDic”)
Glutamate Glu E (“glutEmate”)
Glutamine Gln Q (“Q-tamine”)
Phenylalani Phe F (“Fenylalanine”)
ne
Tyrosine Tyr Y (“tYrosine”)
Tryptophan Trp W (double ring in the
molecule)

26. 4. Letter close to initial letter:
Asx B
Aspartate or
asparagines
Glutamate or Glx Z
glutamine
Lysine Lys K (near L)
Undetermined X
amino acid

28. F. Optical properties of amino acids:
• α-C of each amino acid attached to 4 different
chemical groups
• α-C is chiral or optically active i.e. it has four
different groups attached to the -carbon
(except Gly). The number of optical isomers is
2n, where n is the number of chiral atoms in the
molecule.
• 2 stereoisomers, optical isomers or
enantiomers: D- and L- forms are mirror images
of one another, only L-forms found in human
bodies

31. I. Overview
• 20 amino acids linked
together with peptide
bonds
• 4 organizational levels:
primary, secondary,
tertiary and quaternary

33. Primary Structure

34. • Primary Structure of Proteins
• Sequence of amino acids = primary
structure
• Genetic diseases result from proteins with
abnormal sequences

35. Primary structure: insulin

36. Peptide Bond
• Not broken when
proteins are
denatured
• Prolonged exposure to
acid or base at high
temps is necessary to
break bonds.

38. • 1. Naming the peptide
• a. order of amino acids in a peptide
• Left (N-terminal a.a.) is written first, C-terminal next
• b. Naming of polypeptides
• component a.a. in peptides called moieties or
residues.
• Except C-terminal, all moieties called –yl instead of –
ine –ate, or -ic
• E.g. valylglycylleucine

39. Characteristics of the peptide bond:
• a. Lack of rotation around the bond:
• partial double bond- rigid and planar. bond
between -C and -amino or –CO group is
rotatable
• b. Trans configuration:
• (steric interference in cis position)
• c. Uncharged but polar:
• like all –CONH2 links, peptide bonds do not
protonate between pH 2-12
• only side chains and N- and C- terminals can ionize
• peptide bond is polar (uncharged) and can be
involved in H-bonding.

40. Characteristics of the peptide bond
3. Trans configuration
• minimizes steric hindrance

41. A peptide bond is formed from a condensation reaction (dehydration) involving two
amino acids.
A molecule of H2O is eliminated.

42. Dipeptide formation
H
H H O
O N C C
H
H3N C C O
H CH
O
CH3
H3C CH3
alanine valine
H2O
peptide bond
H O H
O
amino carboxyl
H3N C C N C C
terminus terminus
H O
( amino group) CH3 CH
H3C CH3
alaninylvaline

43. Characteristics of the peptide bond
H O H
O
H3N C C N C C
H O
R1 R2

44. Characteristics of the peptide bond
H O H
O
1. partial double-bond character
H3N C C N C C
• due to resonance H O
R1 R2
O R2 O R2
H H
C C O C C O
C N C C
C N
H3N H H3N H
H O H O
R1 R1
O R2
H
C C O
C C
N
H3N H
H O
R1

45. Characteristics of the peptide bond
2. rigid and planar
• rotation occurs around single bonds but not around double bonds
no rotation around
peptide bond
H O H
O
H3N C C N C C
H O
R1 R2

46. O
O H
C
Rotation around single bonds H
C C
O
C N R2
H3N
H
R1
Because no rotation is possible around O R2
double bonds, the stereochemistry of the
H
peptide bond is fixed. C C O
C N C
H3N H
H O
R1
O R2
R1
C C O
H
C N C
H
H O
NH3

47. Characteristics of the peptide bond
4. Uncharged but polar
• dipole moment exists due to separation of charge
O R2
H
C C O
C C
N
H3N H
H O
R1

48. Characteristics of the peptide bond - summary
• partial double bond character
• rigid and planar
• trans configuration
• uncharged but polar
amide
trans plane
config
O R2
H
C C O
C N C
H3N H
H O
R1

49. B. Determination of the amino acid
composition of a polypeptide
• First, identify and quantify constituent amino acids.
• Pure sample must be used, contamination gives
errors.
• 1. Acid hydrolysis:
• Hydrolyzed by strong acid at 110 C for 24 h
• Peptide bonds cleaved
• Gln & Asn Glu & Asp; Trp mostly destroyed
• Procedure gives composition but not sequence

50. • 2. Chromatography:
• Individual aa’s separated by cation-exchange chromatography
• Anion-exchange resin for -vely charged aa’s
• Eluted from column by buffers of increasing ionic strength and pH
• aa’s separated at different ionic strength and pH
• 3. Quantitative analysis:
• Quantified with ninhydrin purple compd. with amino acids, NH3
and amines (yellow color with imino group of Pro).
• Intensity of color measured in spectrophotometer
• Area under curve proportional to amount of amino acid
• If MW of protein known, no. of residues of each aa known, otherwise,
only ratio of no. of molecules of each amino acid determined.
• Done using amino acid analyzer

52. C. Sequencing of the peptide from its N-terminal
end
• Phenylisothiocyanate –
Edman’s reagent – used to
label N-terminal res under
mildly alkaline conditions.
phenylthiohydantoin (PTH).
• This makes N-terminal
residue peptide bond weak;
break it without breaking
others.
• Above process occurs in a
cycle to sequence peptide
using “sequenator”
• Can be used for
polypeptides of 100 a.a. or
less.

53. O
H2N CH C Lys His Phe Leu Arg COOH
CH3
N C S
N-terminal 1. Labeling
Phenylisothiocyanate
alanine
(Edman’s reagent)
H
O
HN CH C Lys His Phe Leu Arg COOH
S C CH3 labeled peptide
H N 2. Acid
cyclization and expulsion of
hydrolysis
shortened peptide chain
O S
+ C
H2N CH C His Phe Leu Arg COOH N NH
(CH2)4 C CH
O
NH 2 CH3
N-terminal PTH-alanine
lysine

54. Cleavage of peptide into smaller fragments
• occurs before Edman degradation
• necessary if peptide is > 100 amino acids in length
• need to use more than one cleaving agent in order to
determine amino acid sequence
• different enzyme/chemical specificity
• overlap peptide fragments in order to determine original
sequence

55. • 2. Chemical Cleavage:
• Cyanogen bromide cleaves polypeptides on –CO side of methionine
residue
• 3. Overlapping peptides:
• Individual peptides sequenced by Edman’s degradation
• Overlapping peptides help determine sequence
• 4. Multimeric proteins:
• Multiple peptides separated (H-bonds and noncovalent bonds) by urea or
guanidine.HCl
• Disulfide bridges broken with performic acid.

57. Secondary Structure

58. • Secondary structures result from local
arrangement of adjacent amino acids into an
organized 3- dimensional structure.
• H-bonds are key to stabilizing these structures.
Secondary structures include:
• Helical Structures
• Beta Structure (maximally extended primary
sequence)
• Random chain (nonrepetitive)

59. Helix
Left-hand Right-hand
helix helix

60. Intrachain Hydrogen Bonding is important in maintaining secondary protein structure.
Here (in the α helix) the carbonyl oxygen from one amino acid is H-bonded to an alpha
nitrogen of the 4th distant amino acid in the polymer.
Hydrogen
bond

61. • 3.6 residues per turn
• R groups extend outward
helix is disrupted by:
1) P and G
2) large numbers of charged aa’s
3) aa’s with bulky R groups

62. Myoglobin

63. Sheet
• “pleated”
• all peptide bond components involved in H-bonding
• strands visualized as broad arrows
N terminal
C terminal
• may be parallel or antiparallel

64. Sheet

67. -Bend
• function to reverse the direction of polypeptide
chain
• often include charged residues
• stabilized by ionic and/or H-bonds
• usually composed of 4 amino acids including Pro
and Gly

68. Supersecondary structure (motif)
• result from local folding of secondary structures
into small, discrete, commonly-observed aggregates
of secondary structures:
• loop
• corner

69. • extended super secondary structures are known as
domains
• barrel
• twisted sheet

70. Tertiary Structure

71. • Tertiary structure is the 3 dimensional form of a
molecule resulting from distant protein-protein
interactions within the same polypeptide chain
(caused by folding of secondary structures):
Globular proteins are characterized as generally
having:
• a variety of different kinds of secondary structure
• spherical shape
• good water solubility
• a catalytic/regulatory/transport role i.e. a dynamic
metabolic function

72. • IV. Tertiary Structure of Globular Proteins
• Tertiary structure – folding of domains and final
arrangement of domains in protein
• Compact, hydrophobic side chains buried in interior
• Maximum hydrogen bonding of hydrophilic groups
within molecule

73. Fibrous proteins are characterized as generally
having:
• one dominating kind of secondary structure
(i.e. collagen helix in collagen)
• a long narrow rod-like structure
• low water solubility
• a role in determining tissue/cellular structure and
function (e.g. collagen, keratin)

74. Interactions involved in maintaining tertiary structu
-N-H• • •N-
Hydrogen bonds -N-H• • •O-
-O-H• • •O-
-O-H• • •N-
Disulfide bonds 2 cysteine (-SH) cystine (-S-S)-
Hydrophobic interactions
(Van der Waals forces)
Electrostatic interactions (ionic
and/or polar interactions)

75. • Domains
• Fundamental functional and 3-D structural units
of polypeptides
• >200 amino acids 2 or more domains
• folding within domain independent of folding
within other domains
• each domain has characteristics of small,
compact, globular protein

76. • 1. Disulfide bonds:
• formed from –SH groups of two
cysteine residues Cystine
• two Cys may be close by or far
away
• stabilize the protein found in
many secreted proteins
• 2. Hydrophobic interactions:
• interactions between nonpolar
side chains of amino acids in
interior of protein

77. • 3. Hydrogen bonds:
• interactions between
polar side chains
• interactions between
polar side chains and
water enhanced
solubility
• 4. Ionic interactions:
• e.g. Interaction of –COO-
of Asp with NH3+ of Lys

78. Protein folding
• Trial and error process that depends on
• Composition of side chains
• H-bonding
• Disulfide bonds
• Ionic interactions
• To result in most stable or favorable structure
• Chaperones: play a role in folding of proteins during
their synthesis (separate, enhance the rate, protect
residues).

79. Denaturation of Proteins
• Destruction of all but primary structure
• Denaturing agents: heat, organic solvents,
mechanical shearing, heavy metals,
detergents, chaotropic agents
• May be reversible or irreversible
• Loss of biological activity

80. Most proteins do not revert to their original tertiary
structures after denaturation.
Ribonuclease enzyme is an exception.

82. Protein misfolding
• Spontaneous
• Mutation
• Proteolytic cleavage, e.g. accumulation of amyloid
plaques (amyloid-β) in Alzheimer’s.
• Abnomal form of tau accumulation in neufibillary
tangles of Alzheimer’s brain
• Prion disease: Creutzfeldt-Jakob disease – humans
• A protein- as a degenerative agent

83. -Sheet in fibrous (Amyloid) protein
• Amyloid protein deposited in brains of
Alzheimer’s disease patients –
twisted -pleated sheet fibrils with 3-D
structure virtually identical to silk fibrils

84. Prion

85. Quaternary Structure

86. Quaternary structure consists of the association
of multimeric proteins (identical or nonidentical)
held together by one or more of the following
noncovalent interactions:
Hydrogen bonds
Hydrophobic interactions
(Van der Waals forces)
Electrostatic interactions (ionic
and/or polar)

87. Hemoglobin