2. -Amino Acid Sequence
-Protein Conformation
-Levels of Protein Structure
-Primary structure
-Secondary structure
-Tertiary structure
-Quaternary structure
-Classification of Proteins
-Denaturation of Protein
4. Peptides and Proteins
Peptides and proteins are polymers of twenty amino
acids connected to each other by peptide bonds.
Oligopeptide is formed of (2 –10) amino acids:
2 amino acids
3 amino acids
4 amino acids
dipeptide,
tripeptide,
tetrapeptide ….etc.
Polypeptide is formed of more than 10 amino acids.
5. In proteins,
almost all carboxyl and amino groups
are
combined in peptide linkage
and
not available for chemical reaction
(except for hydrogen bond formation).
6. Food Proteins
-Like peptides, proteins are formed from amino acids through
amide linkages.
-Covalently bound hetero constituents can also be incorporated
into proteins. For example, phosphoproteins such as milk casein or
phosvitin of egg yolk contain phosphoric acid esters of serine and
threonine residues.
-The structure of a protein is dependent on the amino acid
sequence (the primary structure) which determines the molecular
conformation (secondary and tertiary structures).
-Proteins sometimes occur as molecular aggregates which are
arranged in an orderly geometric fashion (quaternary structure).
-The sequences and conformations of a large number of proteins
have been elucidated and recorded in several data bases.
7. -Glycoproteins,
such as casein, various
components of egg white
and egg yolk, collagen
from connective tissue
and serum proteins of
some species of fish,
contain one or more
monosaccharide
or
oligosaccharide
units
bound O-glycosidically to
serine,
threonine
or
hydroxylysine
or
Nglycosidically
to
asparagine.
9. 1-Amino Acid Composition, Subunits
-Sequence analysis can only be conducted on a pure protein.
-First, the amino acid composition is determined after acidic hydrolysis.
-The procedure (separation on a single cation-exchange resin column and color
development with ninhydrin reagent) has been standardized and automated
(amino acid analyzers).
-As an alternative to these established methods, the derivatization of amino
acids with the subsequent separation and detection of derivatives is possible
(pre-column derivatization).
Various derivatization reagents can be selected, such as:
• 9-Fluorenylmethylchloroformate (FMOC)
• Phenylisothiocyanate (PITC)
• Dimethylaminoazobenzenesulfonylchloride (DABS-Cl)
• Dimethylaminonaphthalenesulfonylchloride (DANS-Cl)
• 7-Fluoro-4-nitrobenzo-2-oxa-1,3-diazole (NBDF)
• 7-Chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBDCl)
• o-Phthaldialdehyde (OPA)
11. Amino Acid Composition, Subunits
-It is also necessary to know the molecular weight (MW) of the protein.
-MW could be determined by gel column chromatography, ultracentrifugation
or electrophoresis.
-It is necessary to determine whether the protein is a single molecule or
consists of a number of different polypeptide chains (subunits) associated
through disulfide bonds or non-covalent forces.
-Dissociation into subunits can be accomplished by a change in pH, by chemical
modification of the protein, such as succinylation, or with denaturing agents
(urea, guanidine hydrochloride, sodium dodecyl sulfate SDS).
-Disulfide bonds, which are also found in proteins which consist of only one
peptide chain, can be cleaved by oxidation of cystine to cysteic acid or by
reduction to cysteine with subsequent alkylation of thiol group to prevent reoxidation.
-Separation of subunits is achieved by chromatographic or electrophoretic
methods.
12. 2-Terminal Groups
-N-terminal amino acids can be determined by treating a protein
with l-fluoro-2,4-dinitrobenzene (Sanger’s reagent) or 5dimethylaminonaphthalene-1-sulfonyl chloride (dansyl chloride).
-Another possibility is the reaction with cyanate, followed by
elimination of the N-terminal amino acid in the form of hydantoin,
and separation and recovery of the amino acid by cleavage of the
hydantoin .
-The N-terminal amino acid (and the amino acid sequence close to
the N-terminal) is accessible by hydrolysis with aminopeptidase, in
which case it should be remembered that the hydrolysis rate is
dependent on amino acid side chains and that proline residues are
not cleaved.
-A special procedure is required when the N-terminal residue is
acylated (N-formyl- or N-acetyl amino acids).
13. -Determination of C-terminal amino acids is possible via the
hydrazinolysis procedure recommended by Akabori:
-The C-terminal amino acid could be then separated from the
amino acid hydrazides by a cation exchange resin.
14. Terminal Groups
-The C-terminal
enzymatically by
amino
acids
can
be
removed
- Carboxypeptidase A which cleaves amino acids with
aromatic and large aliphatic side chains,
- Carboxypeptidase B which cleaves lysine, arginine and
amino acids with neutral side chains or
- Carboxypeptidase C which cleaves with less specificity
but cleaves proline.
15. 3- Partial Hydrolysis
-Long peptide chains are usually fragmented. The fragments are then analyzed
for amino acid sequences.
-Selective enzymatic cleavage of peptide bonds is accomplished primarily with
Trypsin, which cleaves exclusively Lys-X- and Arg-X-bonds, and
Chymotrypsin, which cleaves peptide bonds with less specificity (Tyr-X, Phe-X,
Trp-X and Leu-X).
-The enzymatic attack can be influenced by modification of protein. For example,
-Acylation of the amino group of lysine limits tryptic hydrolysis to Arg-X,
-Substitution of the SH-group of cysteine residue with an aminoethyl group
introduces a new cleavage position for trypsin into the molecule “pseudolysine
residue”
16. Partial Hydrolysis
-Also suited for the specific
enzymatic hydrolysis of
peptide chains is the
endoproteinase Glu-C from
Staphylococcus aureus. It
cleaves Glu-X bonds as well
as Glu-X plus Asp-X bonds.
-The
most
important
chemical
method
for
selective cleavage uses
cyanogen bromide (BrCN)
to attack Met-X-linkages.
17. Partial Hydrolysis
-Hydrolysis of proteins with strong
acids reveals a difference in the rates
of hydrolysis of peptide bonds
depending on the next amino acid
side chain.
-Bonds involving amino groups of
serine and threonine are particularly
sensitive to hydrolysis.
-This effect is due to N→O-acyl
migration
via
oxazoline
and
subsequent hydrolysis of the ester
bond.
-Hydrolysis of proteins with dilute
acids cleaves aspartyl-X-bonds.
18. Partial Hydrolysis
-Separation of peptide fragments is achieved by gel and ionexchange column chromatography using a volatile buffer as eluent
(pyridine) which can be removed by freeze-drying of the fractions.
-The separation of peptides and proteins by reversed-phase HPLC
has gained great importance, using volatile buffers mixed with
organic, water-soluble solvents as the mobile phase.
-The fragmentation of the protein is performed by different
enzymic and/or chemical techniques, at least by two enzymes of
different specifity.
-The arrangement of the obtained peptides in the same order as
they found in the protein is accomplished with the aid of
overlapping sequences.
19. 4- Sequence Analysis
-The classical method is the Edman degradation reaction.
-It involves stepwise
phenylisothiocyanate.
degradation
of
peptides
with
-The resultant phenylthiohydantoin is identified directly.
-The stepwise reactions are performed in solution or on peptide
bound to a carrier, i. e. to a solid phase.
-Both approaches have been automated (“sequencer”). Carriers
used include resins containing amino groups (e.g., amino
polystyrene) or glass beads treated with amino alkylsiloxane:
20. Sequence Analysis
-The peptides are then attached to the carrier by
carboxyl groups (activation with carbodiimide as
in peptide synthesis) or by amino groups.
-For example, a peptide segment from the
hydrolysis of protein by trypsin has lysine as its
C-terminal amino acid. It is attached to the
carrier with phenylene-diisothiocyanate through
amino groups.
-Mild acidic treatment of the carrier under
conditions of the Edman degradation splits the
first peptide bond.
-The Edman procedure is then performed on
the shortened peptide through second, third and
subsequent repetitive reactions:
22. Protein molecule can be formed of
one or more
polypeptide chains
which may vary in the number
and sequence of amino acid residues.
23. Extended Peptide Chains
-Information about conformation is available
through X-ray crystallographic analysis of
protein crystals and by measuring the distance
between selected protons of the peptide chain
by means of H-NMR spectroscopy in solution.
-X-ray structural analysis of a fully extended
peptide chain reveal the lengths and angles of
bonds
-The peptide bond has partial (40%) double
bond character with electrons shared between
the C-O and C-N bonds.
-The resonance energy is about 83.6 kJ/mole
Structure of an elongated
peptide chain.
24. Levels of Protein Structure
Primary structure
Secondary structure
Tertiary structure
Quaternary structure
25. Primary structure
It is the amino acid sequence of the polypeptide chain
linked by peptide bonds.
It is characteristic for every protein.
All proteins have an
N-terminal end (with a free amino group) and
C-terminal end (with a free carboxyl group).
Polypeptide chain sequence is written according to
the sequence of amino acid residues from the N to C
terminus amino acids.
26.
27. Secondary structure
Is the local spatial arrangement of the polypeptide’s
backbone (peptide bond) atoms without regard to the
conformations of its side chains.
Peptide bonds contain polar amide hydrogen atoms
(with a partial positive charge) and polar carbonyl
oxygen atoms (with a partial negative charge).
This allows hydrogen bonds to form between peptide
bonds in different parts of the chain.
The polypeptide chain can take different shapes or
patterns in different parts of the chain, and these
patterns are called the secondary protein structure.
There are 2 types of secondary structure:
Alpha helix (α-helix)
Beta-pleated sheet (β-pleated sheet).
28. Secondary structure
Alpha helix
• A spiral, compact, rod like structure
• Mostly right handed α-helix, with R
groups protruding outside
• Stabilized by numerous hydrogen bonds
which are formed between carbonyl
oxygen (C=O, hydrogen acceptor) and
peptide nitrogen (NH, hydrogen donor).
• Forms about 100% of fibrous protein
-keratin
-80% of the globular protein; hemoglobin.
29. Alpha helix
Alpha helix is disrupted by:
• Proline: its imino group is not
geometrically compatible with
α- helix.
• Large numbers of bulky amino
acids e.g. tryptophan because of
steric interference.
• Large numbers of branched
amino acids e.g. valine and
isoleucine because of steric
interference.
• Large numbers of acidic and
basic amino acids because they
form ionic bonds or electrically
repel each other.
30. β- PLEATED SHEET
• Almost fully extended and its
surface appear pleated.
• Found in fibrous and globular
protein.
• Formed of 1 or
polypeptide chains.
more
• Stabilized by hydrogen bonds
between peptide bonds.
31. Types of β-PLEATED SHEET
1. Parallel β-pleated sheet: formed
of 2 or more polypeptide chains
running in the same direction (Nterminals are on the same side)
2. Anti-parallel β-pleated sheet:
formed of one or more
polypeptide chains running in
opposite directions (N and C
terminals are alternating).
32.
33. Comparison of -helix and -sheet
-helix
-sheet
Structure
1 polypeptide chain
1 or more polypeptide chains
polypeptide
Coiled
Almost fully extended
Hydrogen
bonds
- Formed
between
2 - Formed between amino acids
peptide bonds of 4 amino
which has no relation in primary
acids apart in the primary
structure.
structure.
- Parallel to the axis of - Perpendicular to the axis of
polypeptide chain.
polypeptide chain.
R groups
- Protrude outside the helix
- Project above and below the
plane of the sheet
36. Tertiary structure
• Is the three dimensional structure
of a single polypeptide chain giving
protein its characteristic shape.
I- Globular proteins (enzymes)
• Approximately spherical shape- water
Soluble.
II- Fibrous proteins (structural
proteins)
•
•
•
•
Rod-like shape
Poor water solubility.
Cross links and bonds in 3ry structure:
S-S bond, Ionic, Hydrophobic interactions
and H-bonding.
Globular
protein
Fibrous
protein
38. Forces that stabilize tertiary structure
These are bonds that form between side
chains of amino acids of the same polypeptide
chain:
1. Disulfide bonds.
2. Hydrophobic interactions.
3. Hydrogen bonds.
4. Ionic interactions.
5. Van der Waal’s forces.
39. Forces that stabilize tertiary structure
Disulfide bonds:
covalent bond between 2 SH groups of 2 cysteine residues forming an S~S
bond of cystine residue.
Hydrophobic interaction:
non covalent bonds between amino acids with non-polar side chains that are
located in the interior of polytpeptide chain away from water.
Hydrogen bonds:
non covalent bond between a hydrogen atom attached to nitrogen or oxygen
and another oxygen or nitrogen atom.
Ionic interaction:
non covalent bonds between negatively charged groups in acidic amino acids
(as carboxilic group in the side chain of aspartate or glutamate) and
positively charged groups in basic amino acids (as amino group in the
side chain of lysine)
Van der Waal’s forces:
non covalent bonds occurring when two adjacent atoms come into closer
distance.
41. Quaternary structure
Many proteins are composed of two or more polypeptide
chains which are loosely associated through noncovalent
interactions (hydrogen bonds, ionic bonds and hydrophobic
interactions).
An individual polypeptide is termed subunit or monomer.
According to the number of subunits, proteins are either:
dimeric (2 subunits),
trimeric (3 subunits),
tetrameric (4 subunits; e.g. HB)
oligomeric (many subunits).
43. Classification of Proteins
Simple
proteins
1. Albumin
2. Globulins
3. Histones
Conjugated
proteins
1.
2.
3.
4.
5.
6.
Phosphoproteins
Glycoproteins
Chromoproteins
Lipoproteins
Nucleoproteins
Metalloproteins
Derived
proteins
Results
from
denaturation
or cleavage of
native proteins by
the action of
acids, alkali or
enzymes.
44. Conjugated Proteins
Proteins can be modified to include other chemical groups
“prosthetic groups” besides amino acids:
Class
Prosthetic group (s)
Example
•Lipoproteins
•Lipids
•VLDL
•Glycoproteins
•Carbohydrates
•Immunoglobulin G
•Phosphoproteins
•Phosphate groups
•Casinogen of milk
•hemoproteins
•Heme (iron porphyrin)
•Hemoglobin
46. Denaturation of Protein
-The term denaturation denotes a reversible or irreversible change
of native conformation (tertiary structure) without cleavage of
covalent bonds (except for disulfide bridges).
The primary structure of the protein is not changed because the
peptide bonds are not affected
Denaturing agents include:
1.
2.
3.
4.
5.
6.
Heat
Changes in pH (concentrated acids or alkali)
Ultraviolet rays
X ray
High salt concentration
Heavy metals.
47. Denaturation
-Denaturation is possible with any treatment that cleaves
hydrogen bridges, ionic or hydrophobic bonds. This can be
accomplished by: changing the temperature, adjusting the pH,
increasing the interface area, or adding organic solvents, salts,
urea, or detergents such as sodium dodecyl sulfate.
-Denaturation is generally reversible when the peptide chain is
stabilized in its unfolded state by the denaturing agent and native
conformation can be re-established after removal of the agent.
-Irreversible denaturation occurs when the unfolded peptide chain
is stabilized by interaction with other chains (as occurs for instance
with egg proteins during boiling). During unfolding reactive groups,
such as thiol groups, that were blocked, may be exposed. Their
participation in the formation of disulfide bonds may also cause an
irreversible denaturation.
49. Effects of Denaturation
-Denaturation destroys the native conformation of protein.
-Denaturation destroys the biologic activity of protein, there is
loss of hormonal, enzymatic and antibody activity.
Applications of protein denaturing
1- Boiling eggs: Change in albumin shape and solubility.
2- Cooking meat: Easily chewable, digestible.
3- Swabbing skin with alcohol (disinfectant):
Denatures/kills bacteria and viruses.
4- HCl in our stomach: denatures proteins and making it easily
digestible by enzymes
- So, eating cooked eggs, meat and liver is more useful to
humans than eating them raw
50. Denaturation of Protein:
Examples in Food
-An aggregation of the peptide
chains caused by the folding of
globular proteins is connected with
reduced solubility or swellability.
-Thus the part of wheat gluten that
is soluble in acetic acid diminishes
as heat stress increases.
-As a result of the reduced rising
capacity of gluten caused by the
pre-treatment, the volume of
bread made of recombined flours
is smaller.
Solubility of gluten
(wheat) in diluted acetic
acid after various forms of
thermal stress
51. Denaturation of Protein:
Examples in Food
-In the case of fibrous proteins, denaturation, through
destruction of the highly ordered structure, generally
leads to increased solubility or rising capacity. One
example is the thermally caused collagen-to-gelatin
conversion, which occurs when meat is cooked.
-The thermal denaturation of the whey proteins βlactoglobulin and α-lactalbumin has been well-studied.
-Denaturation of biologically active proteins is usually
associated with loss of activity. The fact that denatured
proteins are more readily digested by proteolytic
enzymes is also of interest.
