TUMS
Azin Nowrouzi, PhD
1
Proteins
• Most abundant biological macromolecules.
• In all cells and all parts of cells.
• Great variety
– Different kinds
– Different sizes, and properties
– Diversity of biological functions and activities
• 20 precursor molecules are the alphabet
of the language of protein structure.
• These precursor molecules are called
amino acids.
2
What is an amino acid?
3
General structure of  amino acids
General structure of an amino acid
4
Classification of Amino Acid
Amino acid Classification is based on the properties
of the Side Chains or R groups when placed in
water, in particular, their polarity or tendency to
interact with water at biological pH (near ph 7.0):
1. Non polar (Hydrophobic)
2. Polar
I. Neutral amino acids
3. Charged
1. Non polar aliphatic (Gly, Ala, Val, Leu, Ile)
– Basic
– Acidic
2.
3.
4.
5.
Polar, aliphatic (Ser, Thr, Asn, Gln)
Aromatic (Phe, Tyr, Trp)
Sulfur-containing (Cys, Met)
Containing seconcary amino group (pro)
II. Acidic amino acids (Asp, Glu)
III. Basic amino acids (Lys, Arg, His)
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Types of amino acids
6
Other types of amino acids
7
Amino Acids Abbreviations
8
Some facts about amino acids
• The first to be discovered was asparagine, in 1806.
– Asparagine was first found in asparagus.
• The last of the 20 to be found, threonine, was not
identified until 1938.
• Glutamate was found in wheat gluten.
• Tyrosine was first isolated from cheese (its name is
derived from the Greek tyros, “cheese”).
• Glycine (Greek glykos,“sweet”) was so named because
of its sweet taste.
• Histidine has an imidazole group in its side chain.
• Lysine has an amino group in its R.
• Arginine has a Guanidine group in its side chain.
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Side chains with unique properties
10
Diastereomers
• L-threonine is 2S-3R.
11
Disulfide bridge
• Reversible formation of a disulfide bond by the oxidation of two
molecules of cysteine.
• Disulfide bonds between Cys residues stabilize the structures of
many proteins.
12
Properties of Amino Acid Side Chains
The twenty kinds of side chains vary in
1. Size & Shape: Large, bulky aromatic to large
aliphatic to small and very flexible CH3 and H
2. Charge (3 Basic; 2 Acidic)
3. Hydrogen-bonding capacity
4. Chemical Reactivity
5. Polarity (7 polar; 8 non-polar)
6. Hydrophobicity
 Thus, providing any given protein molecule
with functional diversity and versatility.
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Uncommon amino acids in proteins
“post-translational modifications”
14
Nonprotein amino acids
In humans, non-protein amino acids also have biologically-important roles.
• Glycine, gamma-aminobutyric acid and glutamate are
neurotransmitters and many amino acids are used to synthesize other
molecules, for example:
• Tryptophan is a precursor of the neurotransmitter seretonin.
• Glycine is a precursor of porphyrins such as heme.
• Arginine is a precursor of nitric oxide.
• Carnitine, made in the body from the amino acids lysine and
methionine, is used in lipid transport within a cell.
• Ornithine and S-adenosylmethionine are precursors of polyamines.
• Ornithine and Citrulline are key intermediates (metabolites) in the
biosynthesis of arginine and in the urea cycle.
• Homocysteine is an intermediate in S-adenosylmethionine recycling.
Amino acid derivative
Use in industry
Aspartame (aspartyl-phenylalanine-1-methyl ester)
Low-calorie artificial sweetener.
5-HTP (5-hydroxytryptophan)
Treatment for depression and the neurological problems of phenylketonuria.
L-DOPA (L-dihydroxyphenylalanine)
Treatment for Parkinsonism.
Monosodium glutamate
Food additive that enhances flavor. Confers the taste umami.
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Nutritional importance
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Physical and chemical
properties of amino acids
1.
Optical properties of amino acids
Stereoisomerism in -amino acids
–
2.
Ionization of amino acids
–
–
3.
Titration curves
Isoelectric pH (pI)
Chemical reactions of the carboxyl group
–
Include all the reactions of weak acids
•
•
•
•
4.
Chemical reactions of the amino group
–
–
–
–
5.
Salt production with bases
Esters with alcohols
Amides with amines
Decarboxylation
Reaction with dinitrofluorobenzene (DNP-aa)
Amides with acarboxilic acids
Reaction with phenyl isothiocyanate
Reaction with ninhydrin
Separation and analysis of amino acids
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L- and D- configurations
are mirror images
L-amino acid
O
O
C
+
H3N C H
R1
-carbon, C, is a
chiral center.
D-amino acid
O
O
C
+
H C NH3
R1
Mirror plane
18
Stereoisomerism in -amino acids
19
Fischer projection formulas
20
Different forms of amino acids
• When an amino acid is dissolved in
water, it exists in solution as the
dipolar ion, or zwitterion (German for
“hybrid ion”).
• Substances having this dual nature of
acid and base are amphoteric and
are often called ampholytes (from
“amphoteric electrolytes”).
A zwitterion can act as
an acid (proton donor):
A zwitterion can act as a
base (proton acceptor):
21
Amino acids as diprotic acids
• A simple monoamino monocarboxylic amino acid, such as alanine, is a diprotic
acid when fully protonated—it has two
groups, the -COOH group and the -NH3+
group, that can yield protons:
22
Titration of an amino acid
Shown here is the titration curve
of 0.1 M glycine at 25 C.
• The ionic species predominating
at key points in the titration are
shown above the graph.
• The shaded boxes, centered at
about pK1 2.34 and pK2 9.60,
indicate the regions of greatest
buffering power.
Titration Curves Predict the Electric
Charge of Amino Acids:
Glycine has a net negative charge at
any pH above its pI and will thus move
toward the positive electrode (the
anode) when placed in an electric field.
At any pH below its pI, glycine has a
net positive charge and will move
toward the negative electrode (the
cathode).
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Isoelectric pH (pI)
• pI= the point where the charges on a zwitterionic molecule
balance
• Each amino acid has an isoelectric focusing point (pI).
pKa1 = 2.34
Net charge
Net charge
0
-1
pKa2 = 9.60
H
H
O
+
+
H3N
O
C
C
H
OH
H3N
C
H
C
O
-
H2N
H
O
C
C
O
H
Net charge
+1
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-
Titration curve for glutamate
pka1  pKaR
pI 
2
Net charge= +1
Net charge= 0
Net charge= -1
Net charge= -2
• There are three ionizable
groups and, therefore,
three transitions in the
titration.
• The pI corresponds to the
midpoint between the two
transitions that involve the
species with no net
charge.
• These amino-acids have
negative net charge at
neutral pH.
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Titration curve for Histidine
Net charge= +2
Net charge= +1
Net charge= 0
Net charge= -1
pKa2  pKaR
pI 
2
• There are three ionizable
groups and, therefore,
three transitions in the
titration.
• The pI corresponds to the
midpoint between the two
transitions that involve the
species with no net
charge.
• These amino-acids have
positive net charge at
neutral pH.
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pI of lysine
+
H3N
Net charge
Net charge
Net charge
Net charge
+2
+1
0
-1
H
O
C
C
+
OH
H3N
CH 2
H
O
C
C
CH 2
pKa1
O-
H2N
pKa2
H
O
C
C
CH 2
O-
H2N
pKaR
H
O
C
C
CH 2
CH 2
CH 2
CH 2
CH 2
CH 2
CH 2
CH 2
CH 2
CH 2
CH 2
CH 2
NH 3
NH 3
NH 3
NH 2
CH 2
+
pK1 = 2.18
pK2 = 8.95
pKR = 10.5
+
+
pKa2  pKaR
pI 
2
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O-
Typical titratable groups on amino acids
pKa
groups

charge at pH 7.0
COOH
3.5-4.0
-
NH3
9.0-10.8
+
COOH
4.3-4.5
-
NH3
10.5
+
NH
6.0
+
N C NH2
H
NH
12.5
+
8.3
0
10.1
0

H
N
H3C
C SH
H2
C
H2
OH
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Properties of Non-polar
(Hydrophobic) aminoacids
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Properties of Polar (Hydrophilic) amino acids
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Peptides and proteins
• Polymers of amino acids.
• Two amino acids join covalently through a peptide bond.
• Another name for peptide bond is amide bond or linkage.
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Peptide bond formation
water is removed
This is a condensation reaction.
•
1.
2.
3.
•
There are only 3 known ways to make a peptide bond
Chemical abiotic synthesis in the laboratory.
Genetic engineering cloning mechanisms.
Biologically in cells.
Peptide bonds in proteins are quite stable, with an average half-life
(t1/2) of about 7 years under most intracellular conditions.
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Peptides are chains of amino acids
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Chains of amino acids
• Dipeptide: 2 amino acids (aas) joined by 1
peptide bond.
• Tripeptide: 3 aas joined by 2 peptide bonds.
• Tetrapeptide: 4 aas joined together by 3 peptide
bonds.
• Peptapeptide and so forth….
• Oligopeptide: Few aas joined together by
peptide bonds.
• Polypeptide: Many aas joined. Molecular
weights generally below 10000.
• Proteins: Many aas joined. Generally have high
molecular weights.
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Nomenclature
• The pentapeptide serylglycyltyrosylalanylleucine,
or Ser–Gly–Tyr–Ala–Leu.
• Peptides are named beginning with the amino terminal residue,
which by convention is placed at the left.
• The peptide bonds are shaded in yellow; the R groups are in red.
35
Ionization behavior of peptides
amino-terminal (N-terminal) residue
carboxyl-terminal (C-terminal) residue
Alanylglutamylglycyllysine
Like free amino acids, peptides have characteristic titration curves and a
characteristic isoelectric pH (pI) at which they do not move in an electric field. 36
Additional topics
1. Relationship between the activity and size
(Mw) of peptides and proteins.
–
Small peptides have important biological activities
2. Length of polypeptide chains.
–
3.
4.
5.
6.
How to calculate the number of amino acids in a
protein.
Amino Acid Compositions of polypeptides.
Simple and conjugated proteins.
Multisubunit proteins.
Levels of structure in proteins.
37
Peptides have important biological activities
Peptide hormones
 Oxytocin- a nonapeptide
involved in parturition and
lactation.
 Vasopressin-maintains water
balance
 Structurally similar, but
different functions
 Contain disulfide bridges
 covalent linkages
 intramolecular cross-links.

Enkephalins
 Either of two penta-peptides
with opiate & analgesic
activity (involved in pain
control).
 Occur naturally in brain &
have marked affinity for
opiate receptors.
•
Aspartame
 low calorie sweetener
 L-aspartyl-L-phenylalanine
methyl ester.
38
Relationship between the activity and
size (Mw) of peptides and proteins
Some of vertebrate hormones are small peptides:
•
•
•
•
•
Oxytocin (nine amino acid residues), which is secreted by the posterior
pituitary and stimulates uterine contractions.
Bradykinin (nine residues), which inhibits inflammation of tissues.
Thyrotropin-releasing factor (three residues), which is formed in the
hypothalamus and stimulates the release of another hormone,
thyrotropin, from the anterior pituitary gland.
Some mushroom poisons, such as amanitin.
Many antibiotics.
Slightly larger are small polypeptides and oligopeptides:
•
•
•
The pancreatic hormone insulin, which contains two polypeptide chains,
one having 30 amino acidresidues and the other 21.
Glucagon, another pancreatic hormone, has 29 residues; it opposes the
action of insulin.
Corticotropin is a 39-residue hormone of the anterior pituitary gland that
stimulates the adrenal cortex.
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Insulin
•
Regulates glucose uptake.
•
Diabetes is caused by the failure
to produce insulin or the failure
to respond to insulin.
•
•
A 51 amino acid protein.
First protein to have its
sequence determined.
•
Two chains connected by
disulfide bonds.
1. alpha chain of 30 aa’s &
2. beta chain of 21 aa
40
Length of polypeptide chains
•
Lengths vary considerably.
41
How to calculate the number
of amino acids in a protein
• We can calculate the approximate number of amino
• acid residues in a simple protein containing no other
chemical constituents by dividing its molecular weight by
110.
• Although the average molecular weight of the 20 common
amino acids is about 138, the smaller amino acids
predominate in most proteins.
• If we take into account the proportions in which the various
amino acids occur in proteins, the average molecular
weight of protein amino acids is nearer to 128.
• Because a molecule of water (Mr 18) is removed to create
each peptide bond, the average molecular weight of an
amino acid residue in a protein is about 128 -18 = 110.
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Polypeptides Have Characteristic
Amino Acid Compositions
• The 20 common amino
acids almost never
occur in equal amounts
in a protein.
• Some amino acids may
occur only once or not
at all in a given type of
protein; others may
occur in large numbers.
43
Multisubunit proteins
• When two or more polypeptides are associated
noncovalently.
– Hemoglobin, for example, has four polypeptide
subunits: two identical  chains and two identical 
chains, all four held together by noncovalent
interactions.
• A few proteins contain two or more polypeptide
chains linked covalently.
– For example, the two polypeptide chains of insulin are
linked by disulfide bonds. In such cases, the
individual polypeptides are not considered subunits
but are commonly referred to simply as chains.
44
Levels of structure in proteins
45
Simple and conjugated proteins
• Some proteins contain chemical groups other than amino acids.
46
Spectroscopy of amino acids
• Aromatic amino-acids are strong chomophores in the far-uv.
• Only the aromatic amino acids absorb light in the UV region
47
Absorbance can be measured
by UV-spectrophotometer
48
Ninhydrin-detection of amino acids
• Complete hydrolysis for 24 hr at 110 oC in 6 M HCl.
• Amino acids can be detected on the chromatogram by using
ninhydrin. A solution of ninhydrin is sprayed onto the paper and
heated. The amino acids show up as purple spots (proline
appears yellow).
49
Paper chromatograms
2D chromatogram
eluting with a
different solvent
mixture in each
direction.
50
Electrophoresis
• Electrophoresis is a technique that uses the net charge of peptides
(amino acids) as a basis for separation.
 A potential difference is applied across a solid material (e.g. paper
for amino acid analysis) permeated by an electrolyte.
 Anions migrate to the anode and cations to the cathode. The rate of
diffusion is related to the size and net charge. Small highly charged
proteins migrate more quickly.
51
Isoelectric focusing (IEF)
• It is difficult to separate two peptides or proteins of
similar MW if they differ only slightly in their net charge at
a given pH.
 This problem can be overcome by performing the
electrophoresis across a pH gradient – known as
isoelectric focusing.
 As soon as the IEP is reached the proteins carry zero
net charge and sostop migrating.
52
Isoelectric focusing (IEF)
• This technique separates
proteins according to their
isoelectric points.
53
Specific cleavage of polypeptides
1. Proteins larger than 50 aa
are first hydrolyzed into
shorter peptides.
2. Chemical or enzymatic
methods hydrolyze proteins
at specific sites.
3. Peptides are separated by
chromatography
4. Peptides generated by 2 or
more cleavage methods are
each sequenced separately.
5. Sequences of individual
peptides are overlapped
together to deduce the entire
protein sequence
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Protein Sequencing Example
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