PROTEINS

Structure and Function

PEPTIDE BOND FORMATION
-The alpha carboxyl group of one amino acid reacts with alpha amino group of another amino
acid to form a peptide bond or the CO-NH bridge.
AMINO ACIDS ARE LINKED BY PEPTIDE BONDS

1. Dipeptide

2. Tripeptide

3. Tetrapeptide

4. Oligopeptide

5. Polypeptide

6. Proteins
STRUCTURE OF PROTEINS
(ORGANIZATION OF PROTEINS)
STRUCTURE OF PROTEINS(ORGANIZATION OF PROTEINS)
1. PRIMARY STRUCTURE- the sequence of amino acids present in its peptide chain or
chains. The higher levels of organization are decided by the primary structure. Each polypeptide
chain has a unique amino acid sequence decided by the genes. The primary structure is maintained
by the covalent peptide bonds (Figure 4.1A).
1-A. Sequence of amino acids in proteins
Students should have a clear concept of the term
"sequence". See the following example:
Gly - Ala - Val (1)
Gly - Val - Ala (2)
Both the tripeptides shown above contain the
same amino acids; but their sequence is altered.
When the sequence is changed, the peptide is also
different.
Gly-Ala-Val (1) Gly-Val-Ala(2)
STRUCTURE OF PROTEINS(ORGANIZATION OF PROTEINS)
1. PRIMARY STRUCTURE
1-B. Numbering of Amino Acids in Proteins
In all peptides, the amino acid at one end (left side)
has a free amino group called the N-terminal end and
the other end (right side) has a free carboxyl group called
the C-terminal end.

Amino acid residues in polypeptides are named

by changing the suffix "-ine" to "-yl", for example,
Glycine to Glycyl. Thus, peptide bonds formed
by carboxyl group of glycine with amino group
of Alanine, and then carboxyl group of Alanine
with amino group of Valine and is called glycyl-alanyl-
valine and abbreviated as
NH2-Gly-Ala-Val-COOH
or Gly-Ala-Val
or simply as GAV
STRUCTURE OF PROTEINS(ORGANIZATION OF PROTEINS)
1. PRIMARY STRUCTURE
1-C. Branched and Circular Proteins
Generally, the polypeptide chains are linear.
However, branching points in the chains may
be produced by interchain disulphide bridges.
The covalent disulphide bonds between
different polypeptide chains in the same protein
(interchain) or portions of the same polypeptide
chain (intrachain) are also part of the primary pseudopeptide
structure.
Rarely, instead of the alpha COOH group the
gamma carboxyl group of glutamic acid may
enter into peptide bond formation, e.g.
Glutathione (gamma-glutamyl-cysteinyl-glycine)
(Fig.15.19).The term pseudopeptide (or isopeptide) is
used to denote such a peptide bond formed
by carboxyl group, other than that present in
alpha position.
STRUCTURE OF PROTEINS(ORGANIZATION OF PROTEINS)
1. PRIMARY STRUCTURE
1-C. Branched and Circular Proteins
Very rarely, protein may be in a circular form,
e.g. Gramicidin- an antibiotic produced by
Bacillus brevis, contains 10 amino acids. It is
circular and contains D-phenyl alanine (usual
proteins contain only L-amino acids).
STRUCTURE OF PROTEINS(ORGANIZATION OF PROTEINS)
1. PRIMARY STRUCTURE
1-D. Primary Structure of Insulin
Insulin has two polypeptide chains. The A chain (Glycine chain) has 21 amino acids and B (Phenyl
alanine) chain has 30 amino acids. They are held together by two interchain disulphide bonds (Fig.
4.4). A chain 7th cysteine and B chain 7th cysteine are connected. Similarly A chain 20th cysteine and B
chain 19th cysteine are connected. There is another intrachain disulphide bond between 6th and
11th cysteine residues of A chain.
STRUCTURE OF PROTEINS(ORGANIZATION OF PROTEINS)
1. PRIMARY STRUCTURE
1-E. Pro-insulin
Beta cells of pancreas synthesize insulin as
a prohormone. Proinsulin is a single
polypeptide chain with 86 amino acids.
Biologically active insulin (2 chains) is formed by
removal of the central portion of the pro-insulin
before release. The C-peptide (connecting
peptide) is also released into the
circulation (Fig. 4.5).
STRUCTURE OF PROTEINS(ORGANIZATION OF PROTEINS)
1. PRIMARY STRUCTURE
1-F. Primary Structure Determines Biological
Activity
A protein with a specific primary structure,
when put in solution, will automatically form its
natural three dimensional shape. So the higher
levels of organization are dependent on the
primary structure.
Even a single amino acid change
(mutation) in the linear sequence may have
profound biological effects on the function. For
example, in HbA (normal hemoglobin) the 6th
amino acid in the beta chain is glutamic acid; it
is changed to valine in HbS (sickle
cell anemia).
STRUCTURE OF PROTEINS(ORGANIZATION OF PROTEINS)
2. SECONDARY STRUCTURE OF PROTEINS
The term "secondary structure" denotes the
configurational relationship between residues
which are about 3–4 amino acids apart in the
linear sequence. Secondary and tertiary levels
of protein structure are preserved by
noncovalent forces or bonds like hydrogen
bonds, electrostatic bonds, hydrophobic
interactions and van der Waals forces.
STRUCTURE OF PROTEINS(ORGANIZATION OF PROTEINS)
INTERACTIONS RESPONSIBLE FOR SECONDARY AND TERTIARY STRUCUTRES

i. A hydrogen bond is a weak electrostatic

attraction between one electronegative atom
like O or N and a hydrogen atom covalently
linked to a second electronegative atom.
Hydrogen atoms can be donated by -NH
(imidazole, indole, peptide); -OH (serine,
threonine) and -NH2 (arginine, lysine). Hydrogen
accepting groups are COO— (aspartic, glutamic) C=O
(peptide); and S–S (disulphide).
ii. Electrostatic bonds (ionic bonds): Positive
charges are donated by epsilon amino group
of lysine, guanidinium group of arginine and
imidazolium group of histidine. Negative
charges are provided by beta and gamma
carboxyl groups of aspartic and glutamic acids.
STRUCTURE OF PROTEINS(ORGANIZATION OF PROTEINS)
INTERACTIONS RESPONSIBLE FOR SECONDARY AND TERTIARY STRUCUTRES

iii. Hydrophobic bonds are formed by interactions

between nonpolar hydrophobic side
chains by eliminating water molecules. This
serves to hold lipophilic side chains together.
iv. The van der Waals forces are very weak, but
collectively contribute maximum towards the
stability of protein structure.
STRUCTURE OF PROTEINS(ORGANIZATION OF PROTEINS)
2. SECONDARY STRUCTURE OF PROTEINS
2-A. Alpha helix
-resembles a coiled spring, with coil configuration
maintained by hydrogen bonds between amino(-NH)
and carbonyl (-C=O) groups of every 4th amino acid
- all amino acid side chains (R groups) lie outside
the helix, there is not enough room for them in the
interior.
- The alpha-helix is the most common
and stable conformation for a polypeptide
chain. In proteins like hemoglobin and
myoglobin, the alpha-helix is abundant, whereas
it is virtually absent in chymotrypsin.
STRUCTURE OF PROTEINS(ORGANIZATION OF PROTEINS)
2. SECONDARY STRUCTURE OF PROTEINS
2-B. Beta-pleated sheet
-involves amino acid chains that are almost
completely extended. Hydrogen bonds form between
two different side-by-side protein chains (interchain
bonds) or between different parts of a single chain that
folds back on itself (intrachain bonds).
-the term pleated sheets arises from the repeated
zigzag pattern in the structure.
- amino acid side chains are located above and
below the plane of the sheet.
- Adjacent strands in a sheet can run in the same
direction with regard to the amino and carboxy
terminal ends of the polypeptide chain (parallel) or in
opposite direction (anti parallel beta sheet) (Fig. 4.7).
Beta-pleated sheet is the major structural motif in
proteins like silk Fibroin (anti parallel), Flavodoxin
(parallel) and Carbonic anhydrase (both).
STRUCTURE OF PROTEINS(ORGANIZATION OF PROTEINS)
2. SECONDARY STRUCTURE OF PROTEINS

Very few proteins have entirely alpha

helix or beta pleated sheet structures.
Instead, most proteins only have certain
portions of their molecules in these
conformations. The rest of the molecule
assumes a ‘random structure’. It is possible
to have both alpha helix and beta pleated
sheet structures within the same protein.
Only sections of the protein where the
side chains (R groups) are relatively
small can have helical or pleated-sheet
structures.
STRUCTURE OF PROTEINS(ORGANIZATION OF PROTEINS)
2. SECONDARY STRUCTURE OF PROTEINS
2-C. Triple helix or Collagen helix
-It is a triple helical structure found in collagen
-involved 3 coiled polypeptide chains wound around each other about a common axis to give a rope-like
arrangement
STRUCTURE OF PROTEINS(ORGANIZATION OF PROTEINS)
3. TERTIARY STRUCTURE
-is the overall three-dimensional shape that results from the attractive forces between amino acid side
chains (R groups) that are widely separated from each other within a chain.
STRUCTURE OF PROTEINS(ORGANIZATION OF PROTEINS)
4. QUATERNARY STRUCTURE
-involves the associations among separate chains
in an oligomeric proteins-two or more polypeptide chains
that are not covalently bonded to each other.
- Depending on the number of polypeptide
chain, the protein may be termed as monomer
(1 chain), dimer (2 chains), tetramer (4 chains)
and so on. Each polypeptide chain is termed
as subunit or monomer. Homodimer contains
two copies of the same polypeptide chain.
Heterodimer contains two different types of
polypeptides as a functional unit.
- For example, 2 alpha-chains and 2 beta-chains
form the Hemoglobin molecule. Similarly, 2
heavy chains and 2 light chains form one
molecule of immunoglobulin G. Creatine
kinase (CK) is a dimer. Lactate dehydrogenase
(LDH) is a tetramer.
hemoglobin
SEQUENCING OF PROTEINS
1.Separating proteins within a cell and purifying them
The key separation and purification methods depend on two physical properties of the proteins: size and
charge.

a. Separating proteins by size

Methods used to separate proteins by size and
mass include ultrafiltration, ultracentrifugation, and
size exclusion chromatography. Ultrafiltration is a
modification of dialysis in which molecules smaller
than a certain size diffuse through a semipermeable
membrane and larger ones don’t. Ultrafiltration can
separate smaller molecules from larger impurities or
larger molecules from smaller impurities.
SEQUENCING OF PROTEINS
1.Separating proteins within a cell and purifying them
The key separation and purification methods depend on two physical properties of the proteins: size and
charge.

a. Separating proteins by size

In ultracentrifugation, a powerful centrifuge causes heavier molecules to sink faster, which allows their
separation, much as the lighter water is separated from the heavier lettuce in a salad spinner. Ultracentrifugation
can also be used to determine a protein’s molar mass.
SEQUENCING OF PROTEINS
1.Separating proteins within a cell and purifying them
The key separation and purification methods depend on two physical properties of the proteins: size and
charge.

a. Separating proteins by size

In size exclusion chromatography, also known as
molecular sieve chromatography, gel filtration
chromatography, or column chromatography, a solution
passes through a chromatography column filled with
porous beads. Molecules that are too large for the pores
pass straight through. Molecules that may enter the
pores are slowed. The molecules that may enter the
pores undergo separation depending on how easily they
can enter.
SEQUENCING OF PROTEINS
1.Separating proteins within a cell and purifying them
The key separation and purification methods depend on two physical properties of the proteins: size and
charge.

b. Separating proteins by charge

Methods used to separate proteins by charge include solubility, ion exchange chromatography, and
electrophoresis. Each of these methods is pH dependent.

In ion exchange chromatography, the greater the magnitude

of the charge, the slower a protein moves through a column.
This relationship is similar to the ion-exchange process that
occurs in water-softening units.
SEQUENCING OF PROTEINS
Digging into the details: Uncovering a protein’s amino acid sequence
When a pure sample of protein is available, you can begin determining its amino acid sequence in order
to identify the specific protein. The general procedure for doing so, with slight modification, works for other
biochemicals as well.

Step 1: Separating and purifying the polypeptide

chains
If you determine that more than one polypeptide
chain is present in the protein, you need to separate
and purify the chains so you can sequence them
individually. (Because many proteins have only one
polypeptide chain, this step isn’t always necessary.)
Denaturing the protein by disrupting its three-
dimensional structure without breaking the peptide
bonds normally suffices. This can be accomplished
by using extremes in pH. If disulfide linkages are
present between the chains, apply the procedure
outlined in Step 2 to separate the chains for isolation.
SEQUENCING OF PROTEINS
Digging into the details: Uncovering a protein’s amino acid sequence
When a pure sample of protein is available, you can begin determining its amino acid sequence in order
to identify the specific protein. The general procedure for doing so, with slight modification, works for other
biochemicals as well.
Step 2: Slashing intrachain disulfide linkages
Step 2 requires breaking (cleaving) the disulfide
linkages. A simple reduction accomplishes this.
However, the linkages may reform later, so you need
to cleave the linkages and prevent their reformation
via reductive cleavage followed by alkylation.
Oxidative cleavage, where oxidation of the sulfur to
–SO3– occurs, also prevents a reversal of the
process.
SEQUENCING OF PROTEINS
Digging into the details: Uncovering a protein’s amino acid sequence
When a pure sample of protein is available, you can begin determining its amino acid sequence in order
to identify the specific protein. The general procedure for doing so, with slight modification, works for other
biochemicals as well.
Step 3: Determining amino acid concentration of
the chain
Step 3 is easily accomplished using an amino acid
analyzer, an automated instrument that can
determine the amino acid composition of a protein in
less than an hour. The instrument requires less than a
nanomole of protein. The analyzer’s output is the
percentages of each of the amino acids present.
However, this simply identifies the components
present and not their order.
SEQUENCING OF PROTEINS
Digging into the details: Uncovering a protein’s amino acid sequence
When a pure sample of protein is available, you can begin determining its amino acid sequence in order
to identify the specific protein. The general procedure for doing so, with slight modification, works for other
biochemicals as well.
Step 4: Identifying the terminal amino acids
Step 4 not only identifies the terminal amino acids
but also indicates whether more than one chain is
present. A polypeptide chain only has one N-
terminal and one C-terminal amino acid. Therefore,
if more than one N- or C-terminal amino acid is
present, more than one polypeptide chain must be
present.
SEQUENCING OF PROTEINS
Digging into the details: Uncovering a protein’s amino acid sequence
When a pure sample of protein is available, you can begin determining its amino acid sequence in order
to identify the specific protein. The general procedure for doing so, with slight modification, works for other
biochemicals as well.
Step 4: Identifying the terminal amino acids
You can also determine the C-terminal residue by
tagging. The akabori reaction (hydrazinolysis) and
reduction with lithium aluminum hydride
tag the C-terminal residue. You can also selectively
cleave the C-terminal residue using the enzyme
carboxypeptidase, a variety of which are available.
Unfortunately, the enzyme doesn’t stop with one
cleavage; given sufficient time, it proceeds down the
entire polypeptide chain.
SEQUENCING OF PROTEINS
Digging into the details: Uncovering a protein’s amino acid sequence
When a pure sample of protein is available, you can begin determining its amino acid sequence in order
to identify the specific protein. The general procedure for doing so, with slight modification, works for other
biochemicals as well.
Steps 5 and 6: Breaking the chain into smaller Using trypsin gives additional information that the total number of
pieces arginine and lysine residues present is one less than the number of
In Step 5, you cleave the polypeptide into smaller fragments generated. The digestive enzyme chymotrypsin
fragments and determine the amino acid composition preferentially cleaves residues containing aromatic rings (tyrosine,
and sequence of each fragment. Step 6 repeats Step 5 phenylalanine, and tryptophan). It slowly cleaves other residues,
using a different cleavage procedure to give a especially leucine. Clostripain cleaves positively charged amino
different set of fragments. Steps 5 and 6 break the acids, especially arginine. It cleaves lysine more slowly. Fragments
chain into smaller pieces to ease identification. Most with a C-terminal aspartic acid or glutamic acid form from the
of the methods here employ enzymes; however, interaction of staphylococcal protease on a protein in a phosphate
other less-specific methods are useful in some cases. buffer. In the presence of bicarbonate or acetate buffer, only C-
Partial acid hydrolysis randomly cleaves the protein terminal glutamic acid fragments result. A number of less specific
chain into a number of fragments. Trypsin, a enzymes can complete the breakdown of the fragments, including
digestive enzyme, specifically cleaves on the C-side elastase, subtilisin, thermolysin, pepsin,
of arginine or lysine. and papain.
SEQUENCING OF PROTEINS
Digging into the details: Uncovering a protein’s amino acid sequence
When a pure sample of protein is available, you can begin determining its amino acid sequence in order
to identify the specific protein. The general procedure for doing so, with slight modification, works for other
biochemicals as well.
Steps 5 and 6: Breaking the chain into smaller
pieces
Chemical methods of breaking up the fragments
include treatment with cyanogen bromide and
hydroxylamine, and then heating in an acidic
solution. Cyanogen bromide specifically attacks
methionine. Hydroxylamine specifically attacks
asparagine-glycine bonds. If a solution at a pH of 2.5
is heated to 104 degrees Fahrenheit (40 degrees
Celsius), selective cleavage of aspartic
acid-proline bonds occurs.
SEQUENCING OF PROTEINS
Digging into the details: Uncovering a protein’s amino acid sequence
When a pure sample of protein is available, you can begin determining its amino acid sequence in order
to identify the specific protein. The general procedure for doing so, with slight modification, works for other
biochemicals as well.
Steps 5 and 6: Breaking the chain into smaller
pieces
Chemical methods of breaking up the fragments
include treatment with cyanogen bromide and
hydroxylamine, and then heating in an acidic
solution. Cyanogen bromide specifically attacks
methionine. Hydroxylamine specifically attacks
asparagine-glycine bonds. If a solution at a pH of 2.5
is heated to 104 degrees Fahrenheit (40 degrees
Celsius), selective cleavage of aspartic
acid-proline bonds occurs. You can apply the Edman
degradation technique to each of the fragments.
This can simplify the determination of the sequence
of a large protein.
SEQUENCING OF PROTEINS
Digging into the details: Uncovering a protein’s amino acid sequence
When a pure sample of protein is available, you can begin determining its amino acid sequence in order
to identify the specific protein. The general procedure for doing so, with slight modification, works for other
biochemicals as well.
Step 7: Combining information to get the total sequence
Step 7 is where the information from the various procedures comes together.
It’s where you assemble the puzzle using the various parts you’ve found. For
example, look at a simple octapeptide fragment from a protein. This fragment
gave, upon complete hydrolysis, one molecule each of alanine (Ala), aspartic
acid (Asp), glycine (Gly), lysine (Lys), phenylalanine (Phe), and valine (Val), as
well as two molecules of cysteine (Cys). The following fragments were isolated
after partial hydrolysis: Gly-Cys, Phe-Val-Gly, Cys-Asp, Cys-Ala, Lys-Cys, and
Cys-Asp-Lys. Now you match the fragments, deduce the amino acid sequence
in the octapeptide, and write a primary structure for the peptide:
SEQUENCING OF PROTEINS
Digging into the details: Uncovering a protein’s amino acid sequence
When a pure sample of protein is available, you can begin determining its amino acid sequence in order
to identify the specific protein. The general procedure for doing so, with slight modification, works for other
biochemicals as well.
Step 7: Combining information to get the total sequence
Step 7 is where the information from the various procedures comes together.
It’s where you assemble the puzzle using the various parts you’ve found. For
example, look at a simple octapeptide fragment from a protein. This fragment
gave, upon complete hydrolysis, one molecule each of alanine (Ala), aspartic
acid (Asp), glycine (Gly), lysine (Lys), phenylalanine (Phe), and valine (Val), as
well as two molecules of cysteine (Cys). The following fragments were isolated
after partial hydrolysis: Gly-Cys, Phe-Val-Gly, Cys-Asp, Cys-Ala, Lys-Cys, and
Cys-Asp-Lys. Now you match the fragments, deduce the amino acid sequence
in the octapeptide, and write a primary structure for the peptide:
Gly-Cys, Phe-Val-Gly, Cys-Asp, Cys-Ala, Lys-Cys, and
Cys-Asp-Lys.
STRUCTURE OF PROTEINS(DISORGANIZATION)
1. PROTEIN HYDROLYSIS
-when a protein or polypeptide in a solution of strong acid or strong base is heated, the peptide bonds of
the amino acid chain are hydrolyzed and free amino acids are produced. The hydrolysis reaction is the
reverse of the formation for a peptide bond.
Example: hydrolysis of Ala-Gly-Cys under acidic condition
STRUCTURE OF PROTEINS(DISORGANIZATION)
2. PROTEIN DENATURATION
-the “unfolding” of protein
-the partial or complete disorganization of a protein’s characteristic three-dimensional shape as a result of
disruption of its secondary, tertiary and quaternary structural interactions.
-because the biological function of a protein depends on its 3D shape, the result of denaturation is loss of
biological activity.
-for a few small proteins, it is possible to find conditions under which the effects of denaturation is
reversed or the protein is “refolded”- this restoration process is called renaturation.
CLASSIFICATION OF PROTEINS
Proteins fall into two general categories:
✓ Fibrous proteins are found only in animals.
They usually serve as structural entities such as
connective tissue, tendons, and muscle fiber.
They’re normally insoluble in water.
✓ Globular proteins don’t usually serve a
structural function. They can act as transporters,
like hemoglobin, and are often enzymes. They’re
usually water-soluble.
CLASSIFICATION OF PROTEINS
Living organisms use proteins in a number of ways:
✓ Structure: Skin and bone contain collagen, a fibrous protein.
✓ Catalysis: Proteins called enzymes allow reactions to occur in an organism under mild
conditions and with great specificity.
✓ Movement: Proteins make up a large percentage of muscle fiber and help in the movement of
various parts of your body.
✓ Transport: Proteins transport small molecules through an organism. Hemoglobin, the protein
that transports oxygen to the cells, is a transport protein.
✓ Hormones: Hormones that happen to be proteins help regulate cell growth.
✓ Protection: Proteins called antibodies help rid the body of foreign harmful substances.
✓ Storage: Some proteins help store other substances in an organism. For example, iron is
stored in the liver in a complex with the protein ferritin.
✓ Regulation: Proteins help mediate cell responses, such as the protein rhodopsin, found in the
eye and involved in the vision process.
PRECIPITATION REACTIONS OF PROTEINS
1. Salting Out
The extent to which a protein is soluble in water
depends on the number of hydrophilic amino acids
found in the protein. The hydrophilic amino acids
are found on the surface of the protein because
they are able to interact with the polar water
molecules via hydrogen bonds while the non-polar
hydrophobic amino acids are found in the core of
protein. When we begin to add salt such as
sodium chloride or ammonium sulfate into our
aqueous protein solution, the solubility of the
proteins begin to decrease. Eventually, when we
reach to a certain salt concentration value, the
protein will become insoluble and will precipitate
(crystallize) out of the solution. This is known as
salting out.
PRECIPITATION REACTIONS OF PROTEINS
2. Iso-electric Precipitation
Proteins are least soluble at their iso-electric pH. Some proteins are precipitated immediately
when adjusted to their iso-electric pH. The best example is Casein which forms a flocculent precipitate at pH
4.6 and redissolves in highly acidic or alkaline solutions. When milk is curdled, the casein forms the white
curd, because lactic acid produced by the fermentation process lowers the pH to the iso-electric point of
casein.

3. Precipitation by Organic Solvents

When an organic solvent is added to the protein solution, water molecules available for proteins
are reduced, and precipitation occurs. Organic solvents reduce the dielectric constant of the
medium which also favors protein precipitation. Hence, alcohol is a powerful protein precipitating
agent.
PRECIPITATION REACTIONS OF PROTEINS
4. Precipitation by Heavy Metal Ions
In alkaline medium, proteins have net negative charge, or are anions. To such a solution, if salts of
heavy metals are added, positively charged metal ions can complex with protein molecules and metal
proteinates are precipitated. Salts of Copper, Zinc, Lead, Cadmium and Mercury are toxic, because
they tend to precipitate normal proteins of the gastro-intestinal wall. Based on this principle, raw
egg is sometimes used as an antidote for mercury poisoning.

5. Precipitation by Alkaloidal Reagents

Tungstic acid, Phosphotungstic acid, Trichloro acetic acid, Picric acid, Sulphosalicylic acid and
Tannic acid are powerful protein precipitating agents. These acids lower the pH of medium, when
proteins carry net positive charges. These protein cations are complexed with negatively charged ions
to form protein-tungstate, protein-picrate, etc. And thick flocculent precipitate is formed. In clinical
laboratory phospho-tungstic or trichloro acetic acid are usually used for precipitating proteins. Tanning
in leather processing is based on the protein precipitating effect of tannic acid.