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
3.
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)
5.
6.
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
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.
12.
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).
14.
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
16.
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
18.
19.
20.
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).
22.
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
27.
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
29.
30.
31. I. Overview
• 20 amino acids linked
together with peptide
bonds
• 4 organizational levels:
primary, secondary,
tertiary and quaternary
36. Peptide Bond
• Not broken when
proteins are
denatured
• Prolonged exposure to
acid or base at high
temps is necessary to
break bonds.
37.
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.
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
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
51.
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.
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)
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
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
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
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)
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.
81.
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
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)