Chapter 3 The Same 20 Amino Acids
Serve as Building Blocks for All
Proteins in Nature
Homework 2 part 1,Ch,5 problems 2,9,12,16.
1.1 Almost all chemical reactions occurring in living
organisms are catalyzed by enzymes.
1.1.1 Many thousands of enzymes have been
discovered,each catalyzing a different kind of
chemical reaction.
1.1.2 Life would not occur without enzyme
catalysis.
1.1.3 Enzymes are the most varied and most
highly specialized proteins.
1,Proteins are extremely versatile in function
and crucial in virtually all biological processes.
1.2 Many small molecules and ions are transported
by specific proteins.
1.2.1 Some proteins transport molecules from
one organ to another,many exist in the blood
plasma,e.g.,hemoglobin (oxygen),serum albumin
(fatty acids),lipoproteins (lipids),and transferrin
(iron).
1.2.2 Some proteins transport molecules
across plasma membrane or organelle membranes
(glucose,amino acids,nucleotides,chloride,
potassium,sodium ions but not water).
1.3 Some proteins function as nutrient or storage
proteins,For example,many of such kind exist in
plant seeds,animal eggs (Ovalbumin of egg white,
casein of milk),Ferritin in animal tissue function to
store ion (about 4500 ions are stored in the cavity of
each ferritin complex!).
1.4 Some proteins are responsible for the coordinated
motions (contraction,changing shape,moving about),
For example,actin and myosin in muscle and
nonmuscle cells,Tubulin and dynein in eukaryotic
flagella and cilia.
1.5 Some proteins are responsible for mechanical
support (strength and protection)
1.5.1 Collagen in tendons (筋腱 ),cartilage,
and leather.
1.5.2 Elastin in ligaments (韧带 ).
1.5.3 Keratin in hair,fingernails,feathers.
1.5.4 Fibroin in silk fibers and spider webs.
1.5.5 Resilin in wing hinges of some insects.
1.6 Some proteins function to defend the organisms
1.6.1 Immunoglobins in vertebrates
1.6.2 Fibrinogen and thrombin in blood-clotting
1.6.3 Snake venom,bacterial toxins,and toxic
plant proteins (ricin).
1.7 Some proteins help regulate cellular or
physiological activity
1.7.1 Growth factors and transcription factors
regulate cell growth and differentiation
1.7.2 Hormones (e.g.,insulin,growth hormon)
coordinate activities of different cells in multicellular
organisms.
1.8 Some proteins are responsible for the
generation and transmission of nerve impulses.
1.8.1 Rhodopsin responses to light to
generate vision
1.8.2 Acetylcholine receptor are
responsible for transmitting nerve impulses at
synapses
1.9 Many proteins have other functions,
Antifreeze proteins protect the blood of
Antarctic fish from freezing,Special heat stable
proteins in thermophile bacteria.
Chymotrypsin with its active site
Fireflies emit light catalyzed by luciferase with ATP
Erythrocytes contain a large amount of hemoglobins,
the oxygen-transporting protein.
The protein keratin is the chief structural components
of hair,scales,horn,wool,nails and feathers.
2,All natural proteins were found to be built
from a repertoire of 20 standard ?-amino acids
2.1 The earliest studies of proteins focused on the free
amino acids derived from these proteins.
2.1.1 The 1st amino acid (asparagine) was
discovered in 1806 from asparagus (a green vegetable).
2.1.2 The last (threonine) was not identified until
1938!
2.1.3 All the amino acids were given a trivial
(common) name,Glutamate from wheat gluten (sticky),
Tyrosine from cheese (“tyros” in Greek).
2.2 The 20 ?-amino acids share common structural
features.
2.2.1 Each has a carboxyl group and an
amino group (but one has an imino group in proline)
bonded to the same carbon atom,designated as the
?-carbon.
2.2.2 Each has a different side chain (or R
group,R=“Remainder of the molecule”).
2.2.3 The ?-carbons for 19 of them are
asymmetric (or chiral),thus being able to have two
enantiomers,Glycine has no chirality.
In protein chemistry,we use Greek letter nomenclature.
2.3 The two enantiomers of each amino acid defined by
the ?-carbon are designated D- and L- forms (D for
Dextrorotary,L for Levorotary)
2.3.1 The D- and L-forms of amino acids are
named in reference to the absolute configuration of D-
and L- glyceraldehydes (whose structure was orignally
assumed and confirmed by X-ray crystallography later).
2.3.2 Only the L-amino acids have been found in
proteins (D-isomers have been found only in small
peptides of bacteria cell walls and in some peptide
antibiotics).
2.3.3 The correlation of structure (or
configuration) with optical rotation is very complex and
has not been successful to date! (i.e.,the D- and L-signs
do not tell anything about their optical rotation!)
2.4 The amino acids ionize in aqueous solutions.
2.4.1 Crystalline amino acids (in neutral
aqueous solutions) have melting points much
higher than those of other organic molecules of
similar size.
2.4.2 The amino acids ionize to various
states depending on pH values.
2.4.3 The amino acids (of neutral side
chains) exist predominantly as dipolar ions,
known as zwitterions (German for,hybrid
ions”).
2.5 Each amino acid is given a three-letter abbreviation
and a one-letter symbol,They often the first three letter
and the first letter,When there is confusion,an
alternative is used,They must be remembered,(fig.)
2.6 All proteins in all species (from bacteria to human)
are constructed from the same set of 20 amino acids.
2.6.1 All proteins,no matter how different they
are in structure and function,are made from the 20
standard amino acids.
2.6.2 This fundamental alphabet of the protein
language is at least two billion years old.
Stryer’s method,walk from the amino group to the carboxyl
group,the hydrogen atom is on your left,L-Alanine
Align carbon atoms with L-glyceraldehyde,the amino group
is on the left,Fig,5-4
The horizontal bonds project out of the plane of the paper,
the vertical behind.
Lined up by similarity,chiral to chiral,COO to CHO
Gly,G
Ala,A
Val,V
Leu,L
Met,M
Ile,I
Phe,F; Tyr,Y; Trp,W
Ser,S
Thr,T
Cys,C
Pro,P
Asn,N
Gln,Q
Lys,K; Arg,R; His,H
Asp,D; Glu,E
3,The 20 amino acids are usually grouped according
to the properties (mainly polarity) of their R groups
3.1 Six amino acids have nonpolar,aliphatic
(hydrophobic) R groups.
3.1.1 They are Gly,Ala,Val,Leu,Ile,and Pro.
3.1.2 Gly has a hydrogen as its R group,having
minimal steric hindrance.
3.1.3 Pro has an imino group,instead of an
amino group,forming a five-membered ring structure,
being rigid in conformation.
3.1.4 Pro is often found in the bends of
folded protein chains and often present on the
surface of proteins.
3.1.5 In protein structure Gly offers the
most flexibility,while Pro the least!
3.1.6 Ala,Val,Leu,and Ile,have
hydrocarbon R groups,often involved in
hydrophobic interactions.
3.2 Phe,Tyr,and Trp have aromatic R groups
3.2.1 Phe and Tyr both have benzene rings.
3.2.2 Tryptophan has an indole ring.
3.2.3 All three participate in hydrophobic
interactions.
3.2.4 The -OH group in Tyr is an important
functional group in proteins,(phosphorylation,
hydrogen bond,etc)
3.2.5 They are jointly responsible for the light
absorption of proteins at 280 nm
A=Log Io/I = ecl Lambert-Beer’s law
e,extinction coefficient; c,concentration; l,optical
length
3.3 Ser,Thr,Asn,Gln,Cys,and Met have polar,
uncharged R groups.
3.3.1 The R groups are more hydrophilic,
due to the presence of hydroxyl groups,sulfur
atoms,or amide groups.
3.3.2 -SH group of two Cys in proteins can
be oxidized to form a covalent disulfide bond.
3.3.3 Cys and Met often participate in
hydrophobic interactions.
3.4 Asp and Glu have carboxyl in their R groups,
They have net negative charge at pH 7.0,thus usually
named as aspartate and glutamate (conjugate base
names,instead of aspartic acid and glutamic acid,
un-ionized form).
3.5 Arg,Lys,and His have positively charged R
groups at pH 7.0.
3.5.1 Their R groups contain guanidino,amino,
imidazole groups respectively.
3.5.2 The side chain of His can be positively or
uncharged depending on the local environment near
pH 7.0
3.6 The extent of hydrophobicity and hydrophilicity
of the side chains is reflected by their hydropathy
index values.,-” values usually mean hydrophilic,“+”
values hydrophobic.
3.7 Nonstandard amino acids are found in certain
proteins,generally as a result of post-translational
modifications.
3.7.1 These modifications are made after the
standard amino acids have been incorporated into
proteins.
3.7.2 4-Hydroxyglutamate and 5-hydroxylysine
in collagen.
3.7.3 g-carboxyglutamate is found in the blood-
clotting prothrombin (an enzyme).
3.7.4 Desmosine is a covalent linkage made
from four Lys side chains in elastin.
3.7.5 Selenocysteine is found in many enzymes
(having been recognized as the 21st amino acid in
ribosome-mediated protein synthesis!).
3.7.6 Many additional nonstandard amino
acids are found in cells,but not in proteins (e.g.,
ornithine and citrulline,intermediates in amino acid
metabolism).
4,Amino acids,being both weak acids and bases,
have characteristic titration curves and pKa
values
4.1 Amino acids can act both as acids and bases,
The zwitterion form of amino acids are
ampholytes,Amino acids can be diprotic and
triprotic acids.
4.2 Monoamino monocarboxylic ?-amino acids (e.g.,
Gly,Ser,Phe with no ionizable groups) all have
similar two-stage titration curves.
4.2.1 The first stage reflects the deprotonation of
the ?-COOH group (pK1).
4.2.2 The second stage reflects the deprotonation
of the ?-NH 3 + group (pK2).
4.2.3 The pKa value of the ?-COOH is more
than 2.0 units smaller than that of acetic acid (pKa of
4.76),that is,a stronger (weak) acid.
4.2.4 These amino acids have two buffering
power regions.
4.3 Acidic and basic amino acids have three-stage
titration curves,The additional stage is for the
ionizable group on the side chains (pKR).
4.4 There is a specific pH (designated pI) at which an
amino acid has equal positive and negative charge.
4.4.1 An amino acid does not move in an electric
field at its pI,called isoelectric point.
4.4.2 The pI of monoamino monocarboxylic
amino acids reflects a status at which the ?-COOH
group is fully deprotonated,but the ?-NH3+ group has
not yet started deprotonating
pI = (pK1+pK2)/2
4.4.3 The pI point of an acidic amino acid reflects
a status at which the ?-COOH is fully deprotonated,
but the side chain -COOH and the a-NH3+ group have
not yet started deprotonating
pI = (pK1+pKR)/2
4.4.4 The pI point of a basic amino acid reflects a
status at which the ?-COOH and the side chain -NH3+
or -NH+= group have fully deprotonated,but the ?-
NH3+ group not yet deprotonated
pI = (pKR+pK2)/2
4.4.5 The amino acids are positively charged at
pH smaller than their pI values,negatively charged at
pH larger than their pI values.
An acidic amino acid pI=(pK1+pKR)/2
A basic amino acid pI=(pKR+pK2)/2
5,The 20 amino acids can be separated from
each other by ion-exchange chromatography
5.1 Each of the amino acids has a different pI
value,Therefore,each amino acid has a
different net charge at a given pH.
5.2 The variously charged amino acids bind to
charged synthetic resins with various affinities.
5.2.1 When the resin is positively charged,
negatively charged amino acids (or other
anions) will bind,and vice versa.
5.2.2 Amino acids having the same charge
as the resin will not bind.
5.2.3 The positively charged resin is called
anion-exchange resin,The negatively charged
called cation-exchange resin (e.g.,the sulfonated
polystyrene).
5.2.4 The resin (serving as the stationary
phase) is usually packed in a column (providing
the mechanical support and stable fluid flow).
5.3 The bound amino acids can be eluted by running a
pH or salt gradient (serving as the mobile phase).
5.3.1 Amino acids will be eluted out in the order
of their binding affinity (strongly bound ones being
eluted out later).
5.3.2 This way of separating amino acids (or
other charged biomolecules) is called ion-exchange
chromatography.
5.3.3 Chromatography is a method of separating
substances by allowing them to partition between two
phases,one mobile,one stationary (differences in
charge,size,hydrophobic interactions,specific
interactions can be exploited for substance separation
with chromatography).
A.J,Martin and R.L,Synge won the Nobel Prize
in 1952 for inventing chromatography.
5.4 The mobile phase can percolate through the
column at low pressure or high pressure.
5.4.1 To operate under high pressure,
specially designed resins and apparatus (the
pumps and the plumbing system) are needed.
5.4.2 Using high pressure allows better
separation in a much shorter period of time,thus
named High Performance Liquid
Chromatography (HPLC).
5.5 Quantitative estimates of each amino acid
in a mixture can be efficiently carried out
using a fully automated Amino Acid Analyzer.
5.5.1 Addition of sample mixture,
elution of each amino acid,collection and
analysis of fractions,and data recording are
all fully automated!
5.5.2 HPLC (ion exchange) column is
used in the machine.
Cation-
exchange
column
Gel filtration
column
Affinity
chromatography
6,The amino acids can be detected using
various chemical reagents
6.1 The free ?-amino group of any amino acids will
react with ninhydrin to form a purple product.
6.1.1 Detects amino acids nonspecifically.
6.1.2 The imino group of Pro gives a yellow
color.
6.1.3 The concentration/amount (micro gram)
of amino acid can be determined by measuring
optical absorbance (at 440 nm for purple or 550 nm
for yellow).
6.2 Other reagents also react with the ?-amino
group,keeping the R group part of the products
6.2.1 1-Fluoro-2,4-dinitrobenzene and
dabsyl chloride react to form colored derivatives
that are stable under harsh conditions (heating
in 6N HCl at 110?C for 24 hours!)
6.2.2 Fluorescent derivatives,permitting
the detection of nanogram amount of amino
acids.
6.2.3 Identity of amino acids can be
revealed by comparing with a standard.
7,Amino acids covalently join one another to
form peptides
7.1 The ?-carboxyl group of one amino acid joins with
the ?-amino group of another amino acid by a peptide
bond (actually an amide bond)
7.1.1 This is a condensation reaction where a
water molecule is liberated or eliminated.
7.1.2 ?G of the condensation reaction is about 5
kcal/mol,not being able to occur spontaneously (an
endergonic reaction).
7.1.3 The condensation reaction can occur
repeatedly to form oligopeptides (with less than 50 aa),
polypeptides (bwt 50-100 aa),and proteins (longer).
7.2 The peptide chain is directional.
7.2.1 An amino acid unit in a peptide
chain is called a residue.
7.2.2 The end having a free ?-amino
group is called amino-terminal or N-terminal.
7.2.3 The end having a free ?-carboxyl
group is called carboxyl-terminal or C-terminal,
7.2.4 By convention,the N-terminal is
taken as the beginning of the peptide chain,and
put at the left (C-terminal at the right),
Biosynthesis starts from the N-terminal.
7.2.5 The peptide chain consist of a
regularly repeating main chain (or backbone)
and the variable side chains of the residues.
7.2.6 Amino acid residues have an order
or sequence on a peptide.
7.2.7 The 20 amino acids are analogous to
the 26 letters in English; the number of
different peptides made of them is unlimited.
7.3 The size of a peptide can be described by its
total number of residues (e.g.,a pentapeptide,a
octapeptide) or relative molecular mass
(molecular weight).
7.3.1 The mean molecular weight of an
amino acid residue in a peptide is ~110 dalton.
7.3.2 Most natural polypeptide chains
contain between 50 and 2000 amino acid
residues,thus having relative molecular mass
between 5500 daltons and 220,000 daltons,or 5.5
kDa and 220 kDa,respectively.
7.4 Each peptide has a characteristic
titration curve and a isoelectric point (pI).
7.4.1 The titration curve of a peptide
reflect the collective behavior of all the acid-
base groups.
7.4.2 The peptide would not move in
an electric field at its pI,(determined by IEF,
isoelectric focusing gel).
7.4.3 2D gel in proteomics,one
dimension is IEF separation by pI; the other
SDS-PAGE separation by molecular weight.
8,Many short peptides have important biological
activities
8.1 Some short peptides (neuropeptides) act as
neurotransmitters,neurohormones,and
neuromodulators.
8.1.1 These peptides are secreted by the neurons.
8.1.2 The LHRH-like decapeptide (10-residue) act
as a neurotransmitter for the frog sympathetic ganglia.
8.1.3 Thyrotropin-releasing factor (3-residue) is
formed in the hypothalamus and stimulates the release
of thyrotropin from the anterior pituitary gland.
8.1.4 Oxytocin (9-residue) is secreted by the
posterior pituitary and stimulates uterine
contraction.
8.1.5 The opioid peptides (including mainly
enkaphalins,endorphins,and dynorphins) have
been implicated in the control of pain,responses
to stress,and other functions.
8.1.6 Some drugs,like morphine and
heroin,generate their addictive effect by binding
to opioid peptide receptors!
8.2 Some short peptides act as antibiotics
8.2.1 Gramicidin A (15-residue) is a well
studied peptide antibiotic (from Bacillus brevis),
Its structure has been determined.
8.2.2 It contains alternating L- and D-amino
acid residues,
8.2.3 It is not synthesized on ribosomes!
8.2.4 Gramicidin S (10-residue,circular) is
another antibiotic also from Bacillus brevis.
8.2.5 Peptide antibiotics have also been
found in frog skins,neutrophile cells,and insects.
8.3 Many short peptides are used as defensive
poisons.
8.3.1 ?-Amantin (8-residue,circular) in
mushroom is an extremely toxic peptide
(inhibiting RNA polymerases II and III at
picomolar levels!)
8.3.2 Very toxic short peptides are also
found in snake venom,spider.
8.4 Many vertebrate hormones are small polypeptides.
8.4.1 Insulin (51-residue) is produced by the
pancreas and acts to lower blood glucose level,after
food intake.
8.4.2 Glucagon (29-residue) is also produced by
the pancreas and acts to increase the blood glucose
level.
8.4.3 Corticotropin (or adrenocorticotropin,39-
residue) is produced by the anterior pituitary gland
and stimulates the growth of adrenal cortex and
secretion of corticosteroid.
8.4.4 Vasopressin (9-residue) stimulates the
reabsorption of water in the distal tubules of the kidney
(diabetes insipidus patients have deficient vasopression)
8.5 Many such bioactive peptides are present in
exceedingly small amounts (thus difficult to
discover!) and acts at very low concentrations.
Bioactive short peptides can be selected by
making random peptide libraries (through
chemical synthesis,combinatory chemistry,or
phage display),Very little is known about the
receptors of these bioactive peptides,They should
be good potential drug targets.
9,Peptides can be synthesized chemically
9.1 Peptides of up to 150 residues can be synthesized
by automated solid-phase methods mainly invented by
R,Bruce Merrifield (who won the 1984 Nobel Prize in
Chemistry for this).
9.1.1 Amino acids are added stepwise to a
growing peptide chain that is linked to an insoluble
matrix,such as polystyrene beads.
9.1.2 A major advantage is that the desired
product at each stage is bound the insoluble beads with
other chemicals easily filtered and washed away.
9.1.3 The synthesis starts with fixing the C-
terminal amino acid on the insoluble beads through its
?-carboxyl group,This is in the reverse direction of
biosynthesis.
9.1.4 The ?-amino group of the next amino acid to
be added is protected and its carboxyl group activated,
The amino group is protected by the t-butyloxycarbonyl
group (t-boc) and deprotected by CF3COOH,The
carboxyl group is activated by dicyclohexylcarbodiimide
(DCC).
9.1.5 The peptide bond is formed by the free ?-
amino group (deprotected) of the fixed C-terminal
residue attacking the DCC activated ?-carboxyl group of
the free amino acid in solution.
9.1.6 After washing away unreacted free amino
acids and other reagents,step 9.1.4 and 9.1.5 are
repeated,The synthesized peptide is eventually cleaved
off from the resin by adding HF.
9.2 The efficiency of this solid phase synthesis is much
lower than biosynthesis in living organisms.
9.2.1 Synthesizing a 100 amino acid peptide will
take about 4 days to finish with a fully automated
machine with reasonable yield.
9.2.2 The same peptide would be synthesized
with exquisite fidelity in about 5 seconds in a bacterial
cell!
9.3 Peptides having natural activity have been
synthesized chemically,
9.3.1 The complete bovine insulin was first
synthesized and show to be the same as the natural
insulin in China in 1965!
9.3.2 Merrifield also synthesized the interferon
(155 aa) and ribonuclease (124 aa).
Serve as Building Blocks for All
Proteins in Nature
Homework 2 part 1,Ch,5 problems 2,9,12,16.
1.1 Almost all chemical reactions occurring in living
organisms are catalyzed by enzymes.
1.1.1 Many thousands of enzymes have been
discovered,each catalyzing a different kind of
chemical reaction.
1.1.2 Life would not occur without enzyme
catalysis.
1.1.3 Enzymes are the most varied and most
highly specialized proteins.
1,Proteins are extremely versatile in function
and crucial in virtually all biological processes.
1.2 Many small molecules and ions are transported
by specific proteins.
1.2.1 Some proteins transport molecules from
one organ to another,many exist in the blood
plasma,e.g.,hemoglobin (oxygen),serum albumin
(fatty acids),lipoproteins (lipids),and transferrin
(iron).
1.2.2 Some proteins transport molecules
across plasma membrane or organelle membranes
(glucose,amino acids,nucleotides,chloride,
potassium,sodium ions but not water).
1.3 Some proteins function as nutrient or storage
proteins,For example,many of such kind exist in
plant seeds,animal eggs (Ovalbumin of egg white,
casein of milk),Ferritin in animal tissue function to
store ion (about 4500 ions are stored in the cavity of
each ferritin complex!).
1.4 Some proteins are responsible for the coordinated
motions (contraction,changing shape,moving about),
For example,actin and myosin in muscle and
nonmuscle cells,Tubulin and dynein in eukaryotic
flagella and cilia.
1.5 Some proteins are responsible for mechanical
support (strength and protection)
1.5.1 Collagen in tendons (筋腱 ),cartilage,
and leather.
1.5.2 Elastin in ligaments (韧带 ).
1.5.3 Keratin in hair,fingernails,feathers.
1.5.4 Fibroin in silk fibers and spider webs.
1.5.5 Resilin in wing hinges of some insects.
1.6 Some proteins function to defend the organisms
1.6.1 Immunoglobins in vertebrates
1.6.2 Fibrinogen and thrombin in blood-clotting
1.6.3 Snake venom,bacterial toxins,and toxic
plant proteins (ricin).
1.7 Some proteins help regulate cellular or
physiological activity
1.7.1 Growth factors and transcription factors
regulate cell growth and differentiation
1.7.2 Hormones (e.g.,insulin,growth hormon)
coordinate activities of different cells in multicellular
organisms.
1.8 Some proteins are responsible for the
generation and transmission of nerve impulses.
1.8.1 Rhodopsin responses to light to
generate vision
1.8.2 Acetylcholine receptor are
responsible for transmitting nerve impulses at
synapses
1.9 Many proteins have other functions,
Antifreeze proteins protect the blood of
Antarctic fish from freezing,Special heat stable
proteins in thermophile bacteria.
Chymotrypsin with its active site
Fireflies emit light catalyzed by luciferase with ATP
Erythrocytes contain a large amount of hemoglobins,
the oxygen-transporting protein.
The protein keratin is the chief structural components
of hair,scales,horn,wool,nails and feathers.
2,All natural proteins were found to be built
from a repertoire of 20 standard ?-amino acids
2.1 The earliest studies of proteins focused on the free
amino acids derived from these proteins.
2.1.1 The 1st amino acid (asparagine) was
discovered in 1806 from asparagus (a green vegetable).
2.1.2 The last (threonine) was not identified until
1938!
2.1.3 All the amino acids were given a trivial
(common) name,Glutamate from wheat gluten (sticky),
Tyrosine from cheese (“tyros” in Greek).
2.2 The 20 ?-amino acids share common structural
features.
2.2.1 Each has a carboxyl group and an
amino group (but one has an imino group in proline)
bonded to the same carbon atom,designated as the
?-carbon.
2.2.2 Each has a different side chain (or R
group,R=“Remainder of the molecule”).
2.2.3 The ?-carbons for 19 of them are
asymmetric (or chiral),thus being able to have two
enantiomers,Glycine has no chirality.
In protein chemistry,we use Greek letter nomenclature.
2.3 The two enantiomers of each amino acid defined by
the ?-carbon are designated D- and L- forms (D for
Dextrorotary,L for Levorotary)
2.3.1 The D- and L-forms of amino acids are
named in reference to the absolute configuration of D-
and L- glyceraldehydes (whose structure was orignally
assumed and confirmed by X-ray crystallography later).
2.3.2 Only the L-amino acids have been found in
proteins (D-isomers have been found only in small
peptides of bacteria cell walls and in some peptide
antibiotics).
2.3.3 The correlation of structure (or
configuration) with optical rotation is very complex and
has not been successful to date! (i.e.,the D- and L-signs
do not tell anything about their optical rotation!)
2.4 The amino acids ionize in aqueous solutions.
2.4.1 Crystalline amino acids (in neutral
aqueous solutions) have melting points much
higher than those of other organic molecules of
similar size.
2.4.2 The amino acids ionize to various
states depending on pH values.
2.4.3 The amino acids (of neutral side
chains) exist predominantly as dipolar ions,
known as zwitterions (German for,hybrid
ions”).
2.5 Each amino acid is given a three-letter abbreviation
and a one-letter symbol,They often the first three letter
and the first letter,When there is confusion,an
alternative is used,They must be remembered,(fig.)
2.6 All proteins in all species (from bacteria to human)
are constructed from the same set of 20 amino acids.
2.6.1 All proteins,no matter how different they
are in structure and function,are made from the 20
standard amino acids.
2.6.2 This fundamental alphabet of the protein
language is at least two billion years old.
Stryer’s method,walk from the amino group to the carboxyl
group,the hydrogen atom is on your left,L-Alanine
Align carbon atoms with L-glyceraldehyde,the amino group
is on the left,Fig,5-4
The horizontal bonds project out of the plane of the paper,
the vertical behind.
Lined up by similarity,chiral to chiral,COO to CHO
Gly,G
Ala,A
Val,V
Leu,L
Met,M
Ile,I
Phe,F; Tyr,Y; Trp,W
Ser,S
Thr,T
Cys,C
Pro,P
Asn,N
Gln,Q
Lys,K; Arg,R; His,H
Asp,D; Glu,E
3,The 20 amino acids are usually grouped according
to the properties (mainly polarity) of their R groups
3.1 Six amino acids have nonpolar,aliphatic
(hydrophobic) R groups.
3.1.1 They are Gly,Ala,Val,Leu,Ile,and Pro.
3.1.2 Gly has a hydrogen as its R group,having
minimal steric hindrance.
3.1.3 Pro has an imino group,instead of an
amino group,forming a five-membered ring structure,
being rigid in conformation.
3.1.4 Pro is often found in the bends of
folded protein chains and often present on the
surface of proteins.
3.1.5 In protein structure Gly offers the
most flexibility,while Pro the least!
3.1.6 Ala,Val,Leu,and Ile,have
hydrocarbon R groups,often involved in
hydrophobic interactions.
3.2 Phe,Tyr,and Trp have aromatic R groups
3.2.1 Phe and Tyr both have benzene rings.
3.2.2 Tryptophan has an indole ring.
3.2.3 All three participate in hydrophobic
interactions.
3.2.4 The -OH group in Tyr is an important
functional group in proteins,(phosphorylation,
hydrogen bond,etc)
3.2.5 They are jointly responsible for the light
absorption of proteins at 280 nm
A=Log Io/I = ecl Lambert-Beer’s law
e,extinction coefficient; c,concentration; l,optical
length
3.3 Ser,Thr,Asn,Gln,Cys,and Met have polar,
uncharged R groups.
3.3.1 The R groups are more hydrophilic,
due to the presence of hydroxyl groups,sulfur
atoms,or amide groups.
3.3.2 -SH group of two Cys in proteins can
be oxidized to form a covalent disulfide bond.
3.3.3 Cys and Met often participate in
hydrophobic interactions.
3.4 Asp and Glu have carboxyl in their R groups,
They have net negative charge at pH 7.0,thus usually
named as aspartate and glutamate (conjugate base
names,instead of aspartic acid and glutamic acid,
un-ionized form).
3.5 Arg,Lys,and His have positively charged R
groups at pH 7.0.
3.5.1 Their R groups contain guanidino,amino,
imidazole groups respectively.
3.5.2 The side chain of His can be positively or
uncharged depending on the local environment near
pH 7.0
3.6 The extent of hydrophobicity and hydrophilicity
of the side chains is reflected by their hydropathy
index values.,-” values usually mean hydrophilic,“+”
values hydrophobic.
3.7 Nonstandard amino acids are found in certain
proteins,generally as a result of post-translational
modifications.
3.7.1 These modifications are made after the
standard amino acids have been incorporated into
proteins.
3.7.2 4-Hydroxyglutamate and 5-hydroxylysine
in collagen.
3.7.3 g-carboxyglutamate is found in the blood-
clotting prothrombin (an enzyme).
3.7.4 Desmosine is a covalent linkage made
from four Lys side chains in elastin.
3.7.5 Selenocysteine is found in many enzymes
(having been recognized as the 21st amino acid in
ribosome-mediated protein synthesis!).
3.7.6 Many additional nonstandard amino
acids are found in cells,but not in proteins (e.g.,
ornithine and citrulline,intermediates in amino acid
metabolism).
4,Amino acids,being both weak acids and bases,
have characteristic titration curves and pKa
values
4.1 Amino acids can act both as acids and bases,
The zwitterion form of amino acids are
ampholytes,Amino acids can be diprotic and
triprotic acids.
4.2 Monoamino monocarboxylic ?-amino acids (e.g.,
Gly,Ser,Phe with no ionizable groups) all have
similar two-stage titration curves.
4.2.1 The first stage reflects the deprotonation of
the ?-COOH group (pK1).
4.2.2 The second stage reflects the deprotonation
of the ?-NH 3 + group (pK2).
4.2.3 The pKa value of the ?-COOH is more
than 2.0 units smaller than that of acetic acid (pKa of
4.76),that is,a stronger (weak) acid.
4.2.4 These amino acids have two buffering
power regions.
4.3 Acidic and basic amino acids have three-stage
titration curves,The additional stage is for the
ionizable group on the side chains (pKR).
4.4 There is a specific pH (designated pI) at which an
amino acid has equal positive and negative charge.
4.4.1 An amino acid does not move in an electric
field at its pI,called isoelectric point.
4.4.2 The pI of monoamino monocarboxylic
amino acids reflects a status at which the ?-COOH
group is fully deprotonated,but the ?-NH3+ group has
not yet started deprotonating
pI = (pK1+pK2)/2
4.4.3 The pI point of an acidic amino acid reflects
a status at which the ?-COOH is fully deprotonated,
but the side chain -COOH and the a-NH3+ group have
not yet started deprotonating
pI = (pK1+pKR)/2
4.4.4 The pI point of a basic amino acid reflects a
status at which the ?-COOH and the side chain -NH3+
or -NH+= group have fully deprotonated,but the ?-
NH3+ group not yet deprotonated
pI = (pKR+pK2)/2
4.4.5 The amino acids are positively charged at
pH smaller than their pI values,negatively charged at
pH larger than their pI values.
An acidic amino acid pI=(pK1+pKR)/2
A basic amino acid pI=(pKR+pK2)/2
5,The 20 amino acids can be separated from
each other by ion-exchange chromatography
5.1 Each of the amino acids has a different pI
value,Therefore,each amino acid has a
different net charge at a given pH.
5.2 The variously charged amino acids bind to
charged synthetic resins with various affinities.
5.2.1 When the resin is positively charged,
negatively charged amino acids (or other
anions) will bind,and vice versa.
5.2.2 Amino acids having the same charge
as the resin will not bind.
5.2.3 The positively charged resin is called
anion-exchange resin,The negatively charged
called cation-exchange resin (e.g.,the sulfonated
polystyrene).
5.2.4 The resin (serving as the stationary
phase) is usually packed in a column (providing
the mechanical support and stable fluid flow).
5.3 The bound amino acids can be eluted by running a
pH or salt gradient (serving as the mobile phase).
5.3.1 Amino acids will be eluted out in the order
of their binding affinity (strongly bound ones being
eluted out later).
5.3.2 This way of separating amino acids (or
other charged biomolecules) is called ion-exchange
chromatography.
5.3.3 Chromatography is a method of separating
substances by allowing them to partition between two
phases,one mobile,one stationary (differences in
charge,size,hydrophobic interactions,specific
interactions can be exploited for substance separation
with chromatography).
A.J,Martin and R.L,Synge won the Nobel Prize
in 1952 for inventing chromatography.
5.4 The mobile phase can percolate through the
column at low pressure or high pressure.
5.4.1 To operate under high pressure,
specially designed resins and apparatus (the
pumps and the plumbing system) are needed.
5.4.2 Using high pressure allows better
separation in a much shorter period of time,thus
named High Performance Liquid
Chromatography (HPLC).
5.5 Quantitative estimates of each amino acid
in a mixture can be efficiently carried out
using a fully automated Amino Acid Analyzer.
5.5.1 Addition of sample mixture,
elution of each amino acid,collection and
analysis of fractions,and data recording are
all fully automated!
5.5.2 HPLC (ion exchange) column is
used in the machine.
Cation-
exchange
column
Gel filtration
column
Affinity
chromatography
6,The amino acids can be detected using
various chemical reagents
6.1 The free ?-amino group of any amino acids will
react with ninhydrin to form a purple product.
6.1.1 Detects amino acids nonspecifically.
6.1.2 The imino group of Pro gives a yellow
color.
6.1.3 The concentration/amount (micro gram)
of amino acid can be determined by measuring
optical absorbance (at 440 nm for purple or 550 nm
for yellow).
6.2 Other reagents also react with the ?-amino
group,keeping the R group part of the products
6.2.1 1-Fluoro-2,4-dinitrobenzene and
dabsyl chloride react to form colored derivatives
that are stable under harsh conditions (heating
in 6N HCl at 110?C for 24 hours!)
6.2.2 Fluorescent derivatives,permitting
the detection of nanogram amount of amino
acids.
6.2.3 Identity of amino acids can be
revealed by comparing with a standard.
7,Amino acids covalently join one another to
form peptides
7.1 The ?-carboxyl group of one amino acid joins with
the ?-amino group of another amino acid by a peptide
bond (actually an amide bond)
7.1.1 This is a condensation reaction where a
water molecule is liberated or eliminated.
7.1.2 ?G of the condensation reaction is about 5
kcal/mol,not being able to occur spontaneously (an
endergonic reaction).
7.1.3 The condensation reaction can occur
repeatedly to form oligopeptides (with less than 50 aa),
polypeptides (bwt 50-100 aa),and proteins (longer).
7.2 The peptide chain is directional.
7.2.1 An amino acid unit in a peptide
chain is called a residue.
7.2.2 The end having a free ?-amino
group is called amino-terminal or N-terminal.
7.2.3 The end having a free ?-carboxyl
group is called carboxyl-terminal or C-terminal,
7.2.4 By convention,the N-terminal is
taken as the beginning of the peptide chain,and
put at the left (C-terminal at the right),
Biosynthesis starts from the N-terminal.
7.2.5 The peptide chain consist of a
regularly repeating main chain (or backbone)
and the variable side chains of the residues.
7.2.6 Amino acid residues have an order
or sequence on a peptide.
7.2.7 The 20 amino acids are analogous to
the 26 letters in English; the number of
different peptides made of them is unlimited.
7.3 The size of a peptide can be described by its
total number of residues (e.g.,a pentapeptide,a
octapeptide) or relative molecular mass
(molecular weight).
7.3.1 The mean molecular weight of an
amino acid residue in a peptide is ~110 dalton.
7.3.2 Most natural polypeptide chains
contain between 50 and 2000 amino acid
residues,thus having relative molecular mass
between 5500 daltons and 220,000 daltons,or 5.5
kDa and 220 kDa,respectively.
7.4 Each peptide has a characteristic
titration curve and a isoelectric point (pI).
7.4.1 The titration curve of a peptide
reflect the collective behavior of all the acid-
base groups.
7.4.2 The peptide would not move in
an electric field at its pI,(determined by IEF,
isoelectric focusing gel).
7.4.3 2D gel in proteomics,one
dimension is IEF separation by pI; the other
SDS-PAGE separation by molecular weight.
8,Many short peptides have important biological
activities
8.1 Some short peptides (neuropeptides) act as
neurotransmitters,neurohormones,and
neuromodulators.
8.1.1 These peptides are secreted by the neurons.
8.1.2 The LHRH-like decapeptide (10-residue) act
as a neurotransmitter for the frog sympathetic ganglia.
8.1.3 Thyrotropin-releasing factor (3-residue) is
formed in the hypothalamus and stimulates the release
of thyrotropin from the anterior pituitary gland.
8.1.4 Oxytocin (9-residue) is secreted by the
posterior pituitary and stimulates uterine
contraction.
8.1.5 The opioid peptides (including mainly
enkaphalins,endorphins,and dynorphins) have
been implicated in the control of pain,responses
to stress,and other functions.
8.1.6 Some drugs,like morphine and
heroin,generate their addictive effect by binding
to opioid peptide receptors!
8.2 Some short peptides act as antibiotics
8.2.1 Gramicidin A (15-residue) is a well
studied peptide antibiotic (from Bacillus brevis),
Its structure has been determined.
8.2.2 It contains alternating L- and D-amino
acid residues,
8.2.3 It is not synthesized on ribosomes!
8.2.4 Gramicidin S (10-residue,circular) is
another antibiotic also from Bacillus brevis.
8.2.5 Peptide antibiotics have also been
found in frog skins,neutrophile cells,and insects.
8.3 Many short peptides are used as defensive
poisons.
8.3.1 ?-Amantin (8-residue,circular) in
mushroom is an extremely toxic peptide
(inhibiting RNA polymerases II and III at
picomolar levels!)
8.3.2 Very toxic short peptides are also
found in snake venom,spider.
8.4 Many vertebrate hormones are small polypeptides.
8.4.1 Insulin (51-residue) is produced by the
pancreas and acts to lower blood glucose level,after
food intake.
8.4.2 Glucagon (29-residue) is also produced by
the pancreas and acts to increase the blood glucose
level.
8.4.3 Corticotropin (or adrenocorticotropin,39-
residue) is produced by the anterior pituitary gland
and stimulates the growth of adrenal cortex and
secretion of corticosteroid.
8.4.4 Vasopressin (9-residue) stimulates the
reabsorption of water in the distal tubules of the kidney
(diabetes insipidus patients have deficient vasopression)
8.5 Many such bioactive peptides are present in
exceedingly small amounts (thus difficult to
discover!) and acts at very low concentrations.
Bioactive short peptides can be selected by
making random peptide libraries (through
chemical synthesis,combinatory chemistry,or
phage display),Very little is known about the
receptors of these bioactive peptides,They should
be good potential drug targets.
9,Peptides can be synthesized chemically
9.1 Peptides of up to 150 residues can be synthesized
by automated solid-phase methods mainly invented by
R,Bruce Merrifield (who won the 1984 Nobel Prize in
Chemistry for this).
9.1.1 Amino acids are added stepwise to a
growing peptide chain that is linked to an insoluble
matrix,such as polystyrene beads.
9.1.2 A major advantage is that the desired
product at each stage is bound the insoluble beads with
other chemicals easily filtered and washed away.
9.1.3 The synthesis starts with fixing the C-
terminal amino acid on the insoluble beads through its
?-carboxyl group,This is in the reverse direction of
biosynthesis.
9.1.4 The ?-amino group of the next amino acid to
be added is protected and its carboxyl group activated,
The amino group is protected by the t-butyloxycarbonyl
group (t-boc) and deprotected by CF3COOH,The
carboxyl group is activated by dicyclohexylcarbodiimide
(DCC).
9.1.5 The peptide bond is formed by the free ?-
amino group (deprotected) of the fixed C-terminal
residue attacking the DCC activated ?-carboxyl group of
the free amino acid in solution.
9.1.6 After washing away unreacted free amino
acids and other reagents,step 9.1.4 and 9.1.5 are
repeated,The synthesized peptide is eventually cleaved
off from the resin by adding HF.
9.2 The efficiency of this solid phase synthesis is much
lower than biosynthesis in living organisms.
9.2.1 Synthesizing a 100 amino acid peptide will
take about 4 days to finish with a fully automated
machine with reasonable yield.
9.2.2 The same peptide would be synthesized
with exquisite fidelity in about 5 seconds in a bacterial
cell!
9.3 Peptides having natural activity have been
synthesized chemically,
9.3.1 The complete bovine insulin was first
synthesized and show to be the same as the natural
insulin in China in 1965!
9.3.2 Merrifield also synthesized the interferon
(155 aa) and ribonuclease (124 aa).
