Download amino acids

TUMS
Azin Nowrouzi, PhD
1
Physical and chemical
properties of amino acids
1.
Optical properties of amino acids
Stereoisomerism in -amino acids, D and L isomers
–
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
Decarboxylation
Amides with amines
Amides with carboxilic acids
Deamination
Reaction with ninhydrin
Reaction with dinitrofluorobenzene (DNP-aa)
Reaction with phenyl isothiocyanate
Separation and analysis of amino acids
2
Examples of decarboxylation
• Formation of Serotonin from tryptophan.
• Formation of -aminobutyric acid (GABA) from
glutamate.
• Formation of neurotransmitters, such as
norepinephrine, dopamine, histamine in the
nervous system.
3
Deamination
• Deamination takes place in the liver.
• It is the process by which amino acids are broken down.
• The amino group is removed from the amino acid and
converted to ammonia.
• The rest of the amino acid is made up of mostly carbon
and hydrogen, and is recycled or oxidized for energy.
• Ammonia is toxic to the human system, and enzymes
convert it to urea or uric acid in the urea cycle.
• Urea and uric acid can safely diffuse into the blood and
then be excreted in urine.
4
Nutritional importance
Essential
Nonessential
Isoleucine
Alanine
Leucine
asparagine
Lysine
Aspartate
Methionine
Cysteine*
Phenylalanine
Glutamate
Threonine
Glutamine*
Tryptophan
Glycine*
Valine
Proline*
Arginine*
Serine*
Histidine*
Tyrosine*
(*) Essential only in certain cases.
5
3- Chemical reactions of the carboxyl
and the amino groups
(Amide or peptide bond formation)
• Polymers of amino acids.
• Two amino acids join covalently through a peptide bond.
• Another name for peptide bond is amide bond or linkage.
6
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.
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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.
11
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
Decarboxylation
Amides with amines
Amides with carboxilic acids
Reaction with ninhydrin (detection and measurement of concentration).
Reaction with dinitrofluorobenzene (DNP-aa) (detection of first amino
acid in a chain and others that have side chains with amino groups)
Reaction with phenyl isothiocyanate (protein sequencing).
Separation and analysis of amino acids
12
Causes of polypeptide diversity
1.
How is the size (Mw) peptides and proteins related to
their activities?
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•
2.
Some small peptides have important biological activities (see next
two slides for examples).
Sometimes larger peptides are needed for certain activities (insulin,
glucagon and corticotropin for example).
How variable are the length of polypeptide chains?
•
•
The length varies considerably.
How to calculate the number of amino acids in a protein.
3.
Amino Acid Compositions of polypeptides.
4.
Simple and conjugated proteins.
5.
Multisubunit proteins.
13
Some peptides with 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.
14
Relationship between the activity and
size (Mw) of peptides and proteins
Some other vertebrate hormones that are small peptides:
• 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
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Length of polypeptide chains
• Lengths vary considerably.
17
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.
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Simple and conjugated proteins
• Some proteins contain chemical groups other than amino acids.
20
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.
21
Levels of structure in proteins
22
Levels of Protein Structure
Rigidity of the
peptide bond
•
-carbons of adjacent amino acids are
separated by three covalent bonds
C-C-N-C
•
Tetrahedral angles:
N-C bond is labeled 
(phi).
C-C bond is labeled  (psi).
Secondary
structures
Primary
structure
•
Amino acid
sequence
•
Repetitive structures
•-Helix
•-Sheet
•parallel
•antiparallel
Non-repetitive structures
•-turn
Tertiary
structure
Quaternary
structure
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Primary structure
• A description of all
covalent bonds (mainly
peptide and disulfide
bonds) linking amino acid
residues in a polypeptide
chain.
• The linear sequence of
amino acids within a
peptide
• Written from NC,
– either in three-letter code,
– or, more often, in one-letter
code.
• Example: Glu-Gly-Ala-Lys or
EGAK
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Insulin’s primary structure
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The planar peptide group
• Each peptide bond has some double-bond character due to resonance.
• Peptide bonds cannot rotate.
• Peptide bond is rigid.
• The six atoms of the peptide group lie in a single plane, with the
oxygen atom of the carbonyl group and the hydrogen atom of the amide
nitrogen trans to each other.
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•
The carbons of adjacent amino acid residues are separated by three
covalent bonds, arranged as C-C-N-C.
•
The N-C and C-C bonds can rotate, with bond angles designated  and
, respectively.
•
The peptide C-N bond is not free to rotate.
•
Other single bonds in the backbone may also be rotationally hindered,
depending on the size and charge of the R groups.
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Steric hindrance
• By convention, both  and
 are defined as 0 when
the two peptide bonds
flanking that carbon are in
the same plane.
• In a protein, this
conformation is prohibited
by steric overlap between
an -carbonyl oxygen and
an -amino hydrogen atom.
• To illustrate the bonds
between atoms, the balls
representing each atom are
smaller than the van der
Waals radii for
• this scale. 1 Å 0.1 nm.
28
Ramachandran plot for L-Ala residues
• The areas shaded dark
blue reflect conformations
that involve no steric
overlap and thus are fully
allowed.
• Medium blue indicates
conformations allowed at
the extreme limits for
unfavorable atomic
contacts.
• The lightest blue area
reflects conformations that
are permissible if a little
flexibility is allowed in the
bond angles.
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Human PCNA
30
Four models of the helix
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-Helix
• H-bonds are inside the chain
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Description of -helix
• The polypeptide backbone is tightly wound
around an imaginary axis drawn longitudinally
through the middle of the helix.
• The R groups of the amino acid residues
protrude outward from the helical backbone.
• The amino acid residues in an -helix have
conformations with  = -40 to -50 and  = -60.
• Each helical turn includes 3.6 amino acid
residues.
• About one-fourth of all amino acid residues in
polypeptides are found in -helices.
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Stabilization of -helix
• The structure is stabilized by a hydrogen bond between
the hydrogen atom attached to the electronegative
nitrogen atom of a peptide linkage (amino acid n) and
the electronegative carbonyl oxygen atom of the fourth
amino acid (amino acid n+4) on the amino-terminal side
of that peptide bond.
• Each successive turn of the -helix is held to adjacent
turns by three to four hydrogen bonds.
• All the hydrogen bonds combined give the entire helical
structure considerable stability.
• Naturally occurring L-amino acids can form either rightor left-handed helices, but extended left-handed helices
have not been observed in proteins.
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Factors affecting stability
1. Electrostatic repulsion (or attraction) between successive amino acid residues
with charged R groups.
2. Bulkiness of adjacent R groups.
3. The interactions between R groups spaced three (or four) residues apart.
4. Presence of Pro or Gly residues.
– Proline is only rarely found within an helix
•
In proline, the nitrogen atom is part of a rigid ring and rotation about the N-C bond is not
possible. Thus, a Pro residue introduces a destabilizing kink in an helix.
•
In addition, the nitrogen atom of a Pro residue in peptide linkage has no substituent hydrogen
to participate in hydrogen bonds with other residues.
– Glycine occurs infrequently in helices for a different reason
•
•
It has more conformational flexibility than the other amino acid residues.
Polymers of glycine tend to take up coiled structures quite different from an -helix.
5. Interaction between amino acid residues at the ends of the helical segment and
the electric dipole inherent to the helix.
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Helix dipole
• A net dipole extends along the
helix that increases with helix
length.
• Negatively charged amino acids
are often found near the amino
terminus of the helical segment,
where they have a stabilizing
interaction with the positive charge
of the helix dipole.
• A positively charged amino acid at
the aminoterminal end is
destabilizing.
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Fully extended chains
can form -sheets
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Types of -sheets
38
- pleated sheet
• H-bonds are
between chains.
•  = 140°
•  = -120°.
39
Two Types of -Pleated Sheets
40
The  conformation of
polypeptide chains
41
42
43
44
Non-repetitive secondary structures
1.
2.
3.
4.
Turns
Connections
Loops
Coils or random coils
•
These are well ordered but non repeating
configurations.
45
 turns
• type I turns occur more than twice as frequently as type II.
• Type II turns always have Gly as the third residue.
46
Tertiary structure
•
Amino acids that are far apart in the
polypeptide sequence and that reside in
different types of secondary structure may
interact within the completely folded
structure of a protein.
•
The location of bends (including turns) in
the polypeptide chain and the direction and
angle of these bends are determined by the
number and location of specific bendproducing residues, such as Pro, Thr, Ser,
and Gly.
•
Interacting segments of polypeptide chains
are held in their characteristic tertiary
positions by different kinds of weak bonding
interactions (and sometimes by covalent
bonds such as disulfide cross-links)
between the segments.
Leptin
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Stabilization of tertiary Structure
• overall threedimensional
arrangement of all
atoms in a protein is
referred to as the
protein’s tertiary
structure.
• Stabilized primarily
through weak
bonds.
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Folding of a polypeptide chain
49
Three-dimensional structures
of some small proteins
Myoglobin
PDB ID 1MBO
Cytochrome c
PDB ID 1CCR
Lysozyme
PDB ID 3LYM
RibonucleasePD
B ID 3RN3
• PDB; www.rcsb.org/pdb
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Interactions stabilizing tertiary structure
• Specific overall shape of a protein
• Cross links between R groups of amino
acids in chain
51
Domains
• When molecular weight is larger than 20000.
• The ratio of surface area to volume is small.
• A protein with multiple domains may appear to
have a distinct globular lobe for each domain.
52
Example
• Crystal structure of the
heterodimeric enzyme
Rab Geranylgeranly
Transferase.
• It is a dimer of a alpha
(blue, red, yellow) and
a beta subunit
(orange).
• The alpha subunit is a
multi domain protein.
53
Quaternary structure
• Protein Quaternary Structures Range from Simple Dimers to
Large Complexes:
• A multisubunit protein is also referred to as a multimer.
• Multimeric proteins can have from two to hundreds of subunits.
• A multimer with just a few subunits is often called an oligomer.
• The repeating structural unit in such a multimeric protein, whether it is
a single subunit or a group of subunits, is called a protomer.
• The first oligomeric protein for which the three dimensional structure
was determined was hemoglobin (Mr 64,500), which contains four
polypeptide chains.
54
Viral capsids
Poliovirus
Tobacco mosaic virus (TMV)
consists of cylindrical coat of 2130
identical subunits enclosing a long
RNA molecule of 6400 nucleotides.
• Supramolecular structures are formed by assembly of
macromolecues and their stepwise joining by nonconvalent bonds.
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Denaturation and Renaturation
•
•
A loss of three-dimensional structure sufficient
to cause loss of function is called
denaturation.
Denaturing agents include:
1. Heat
2. pH
3. Certain miscible organic solvents such as alcohol
or acetone.
4. Certain solutes such as urea and guanidine
hydrochloride.
5. Detergents, such as sodium dodecyl sulfate (SDS).
•
Denaturation of some proteins is reversible.
– This process is called renaturation
57
Renaturation of unfolded, denatured ribonuclease
• Urea is used to
denature ribonuclease,
and mercaptoethanol
(HOCH2CH2SH) to
reduce and thus
cleave the disulfide
bonds to yield eight
Cys residues.
• Renaturation involves
reestablishment of the
correct disulfide crosslinks.
58
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
59
Absorbance can be measured
by UV-spectrophotometer
60
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).
61
Paper chromatograms
2D chromatogram
eluting with a
different solvent
mixture in each
direction.
62
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.
63
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.
64
Isoelectric focusing (IEF)
• This technique separates
proteins according to their
isoelectric points.
65
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
66
Protein Sequencing Example
67
Protein structure
1. 3d structure
of a protein is
determined by
Its amino acid
sequence.
2. The function
of a protein
depends on its
structure.
4. Specific structures
of proteins are stabilized
by weak non covalent
interactions.
3. Isolated proteins
exist in one or a small
number of stable
structural forms.
5. Common structural
patterns can help us
organize our
understanding
of protein architecture.
68
Types of proteins
• In considering these higher levels of structure, it is useful
to classify proteins into two major groups:
• Fibrous proteins, having polypeptide chains arranged
in long strands or sheets.
– Fibrous proteins usually consist largely of a single type of
secondary structure.
– Provide support, shape, and external protection to vertebrates
– -Keratin
– Collagen
• Globular proteins, having polypeptide chains folded
into a spherical or globular shape.
– Globular proteins often contain several types of secondary
structure
– Most enzymes and regulatory proteins are globular proteins
– Myoglobin
69