Protein Introduction

Protein 1
Md. Saiful Islam
B.Pharm, M.Pharm (PCP)
North South University
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Proteins
Proteins belong to a class of organic compound called polyamides.
Polyamides are polymers where the monomer units are held together
by amide groups. The monomer units in proteins are called -amino
acids. The amide group -CO-NH- joining two -amino acids is often
called a peptide bond, so scientists sometimes call single polymer
chains made from -amino acids as polypeptides. Proteins can be
made from a single polypeptide chain or from several polypeptide
chains joined together.
Only twenty -amino acids are commonly found in proteins; so how
can they form such a wide variety of polymers with such different
properties and functions? The simplest way to appreciate this is to
imagine that the alpha-amino acids are like letters in the alphabet
and that proteins are like words. Nature makes many different
proteins from twenty -amino acids, in the same way that we can
make a dictionary full of words from just twenty six letters. some
protein chains contain several hundred -amino acid units.

Proteins make up about 15% of the mass of the average person. Protein
molecules are essential to us in an enormous variety of different ways.
Much of the fabric of our body is constructed from protein molecules.
Muscle, cartilage, ligaments, skin and hair - these are all mainly protein
materials.
In addition to these large scale structures that hold us together,
smaller protein molecules play a vital role in keeping our body working
properly. Hemoglobin, hormones, antibodies , and enzymes are all
examples of these less obvious proteins.
Some micro-organisms in the soil can fix molecular nitrogen from the
air into water-soluble ions such as nitrate (NO3-) and ammonium
(NH4+). Plants can then absorb this inorganic nitrogen and use it to
make their proteins and other nitrogen containing molecules. Animals
feed off the plants and recycle the -amino acid building blocks into
their own protein. Excretion, death and decay ensure that the
nitrogen compounds produced can be re-used by other organisms. This
nitrogen cycle is a well known example of how the elements of life are
used over and over again.

Functions:
1. Required for growth and repair of body tissues
2. Synthesis of enzymes, hormones, and many immune molecules
3. Essential body processes such as water balancing, nutrient
transport and muscle contractions requires protein,
4. Protein is also serves as a source of energy,
5. Protein helps to keep skin, nails and hair healthy
6. Protein is absolutely crucial for overall good health
Protein Requirements:
Children: 2 - 1.5g/kg body weight
Adolescent (10-18yrs): 1g/kg body weight
Adults: 0.8g/kg body weight
Athlets: 1.0g/kg body weight
Pregnant/lactating mother: +30g/day

Protein Turnover
Most of the proteins in the body are constantly being synthesized
and then degraded, abnormal and unneeded proteins are removed.
In healthy adults the total amount of protein in the body remains
constant, because the rate of protein synthesis is just sufficient to
replace the protein that is degraded, this process called protein
turnover. The amount of protein turn over in an adult person is about
300 to 400g in each day.
The rate of protein turnover varies widely for individual proteins.
Short-lived proteins (eg, regulatory proteins, misfolded proteins) are
rapidly degraded, their half-lives are few minutes or hours. Long-
lived proteins have half-lives of days to weeks, it constitute the
majority of protein in the cell. Structural proteins, such as collagen
are metabolically stable, and have half-lives measured in months or
years.

Protein degradation: There are two ways
Through ubiquitin pathway: Endogenous proteins are degraded in this way
(80-90%)
Through lysosomal enzymes: Extracellular proteins (endocytosis, cell
surface membrane proteins) are degraded (10-20%)
Ubiquitin Pathway
Ubiquitin, a small globular protein, binds to the ubiquitin domain of the
cellular protein. The consecutive addition of ubiquitin generates a
polyubiquitin chain. Proteins tagged with polyubiquitin are then recognized by
a complex called proteasome which degraded proteins to amino acids.
N-terminal amino acid of a protein influences the half-life of a protein. For
example, proteins that have serine as N-terminal amino acid have half-life
more than 20 hours. Proteins that have Aspartic acid as N-terminal amino
acid have half-life only three minutes. Furthermore, proteins rich in proline,
glutamic acid, serine and threonine are rapidly degraded.

Classification of Proteins: According to biological role, 8 types
1. Enzymes: Most highly specialized proteins are those with catalytic
activity and are called enzymes. To date over 7000 different
enzymes have been discovered in different form of life.
2. Transport proteins: Proteins which carry specific molecules or ions
from one organ to another. Hb of RBC binds with oxygen and
carries to the peripheral tissues from lungs. Nutrients like
glucose, amino acids, lipids are transported across the membrane
into cells using different kind of proteins.
3. Storage proteins: Many plants stored proteins for growth of
embryonic plant, eg, seed proteins of wheat, corn and rice.
Ferritin of animal tissues stores iron, ovalbumin in egg, casein
protein of milk

4. Contractile and motile proteins: This type of proteins have ability
to contract, to change shape or move. Actin, myosin functioning in
the contractile system of skeletal muscle. Tubulin, component of
microtubules are important for flagella and cilia, used for
movement.
5. Structural proteins: Many proteins serve as supporting to give
biological structures, strength or protections. Collagen for tendon
and cartilage, elastin for ligaments, keratin for hair, fingernails and
feathers, fibroin for spider webs,
6. Defense proteins: Many proteins defend organisms against invasion
of other species to protect the cell from injury. Immunoglobulins or
antibodies are specialized proteins which can neutralize invading
bacteria, viruses or foreign proteins from another species.
Fibrinogen and thrombin are blood clotting proteins that prevent
loss of blood during injury. Snake venom, bacterial toxin, toxin plant
proteins are also function in defense.

7. Regulatory proteins: Some proteins help to regulate cellular or
physiological activity. Hormones such as insulin which regulate
sugar metabolism. Growth hormone/parathyroid hormone
regulates calcium and phosphate transport.
8. Other proteins (not easy to classify): Monellin—African plant
protein, sweet taste, nonfunctioning character. Antifreeze
protein, contains in Antarctic fish, Resilin – perfect elastic
properties.
Classification according to shape:
Globular proteins: polypeptide chains are tightly folded into globular
shape, eg- enzymes
Fibrous proteins: long stringy molecules, a-keratin – hair/wool,
fibroin – silk, collagen – tendons.

Amino acids:
Amino acids are molecules containing an amine group, a carboxylic acid
group and a side chain that varies between different amino acids.
These molecules contain the key elements of carbon, hydrogen, oxygen,
and nitrogen. These molecules are particularly important in
biochemistry, where this term refers to -amino acids with the
general formula H2N-CHRCOOH, where R is an organic substituent. In
an alpha amino acid, the amino and carboxylate groups are attached to
the same carbon atom, which is called the α–carbon.
The general structure of an alpha amino acid.

Lysine with the carbon
atoms in side chain labeled
Numbering of Carbon atoms in
Amino acids: The carbon atom
next to the carbonyl group is called
the α–carbon and amino acids with a
side chain bonded to this carbon are
referred to as alpha amino acids.
These are the most common form
found in nature.
In amino acids that have a carbon
chain attached to the α–carbon (such
as lysine, shown to the right), the
carbons are labeled in order as α, β,
γ, δ, and so on

An amino acid in its
(1) Unionized and
(2) Zwitterionic forms
Zwitterions
Amino acids have both amine and carboxylic acid functional groups
and are therefore both an acid and a base at the same time. At a
certain pH when an amino acid has no net charge because of the
number of protonated ammonium groups (positive charges) and
deprotonated carboxylate groups (negative charges) are equal is
known as the isoelectric point and express as pI. All the amino acids
have different isoelectric points. The ions produced at the
isoelectric point have both positive and negative charges and are
known as a zwitterion, which comes from the German word Zwitter
meaning "hybrid". Amino acids can exist as zwitterions in solids and in
polar solutions such as water, but not in the gas phase.

Non-protein functions of amino acids
In humans, non-protein amino acids also have important roles as
metabolic intermediates, such as in the biosynthesis of the
neurotransmitter, gamma-aminobutyric acid.
Ornithine and S-adenosylmethionine are precursors of
polyamines.
Many amino acids are used to synthesize other molecules, for
example:
•Tryptophan is a precursor of the neurotransmitter serotonin.
•Glycine is a precursor of porphyrins such as heme.
•Arginine is a precursor of nitric oxide.
•Aspartate, glycine and glutamine are precursors of nucleotides.

Classification of amino acids: On the basis of R-groups
amino acids are classified into 4 different groups
Nonpolar (hydrophobic) R groups: 8 amino acids
The R group in this class of amino acids are hydrocarbon in nature
and thus hydrophobic.
This group includes five amino acids with aliphatic R groups:
alanine, valine, leucine, isoleucine and proline,
Two with aromatic rings: Phenylalanine, tryptophan and
One containing sulfur: Methionine.

Nonpolar (hydrophobic) R groups: 8 amino acids

Polar but uncharged R groups: The R groups of these
amino acids are more
soluble in water, ie, more
hydrophilic than those of
the nonpolar amino acids
because they contain
functional groups that
forms hydrogen bonds
with water.
The polarity of ser, Thr
and Tyr is contributed
by their hydroxyl
groups, Asn and Gln by
their amide groups, Cys
by its sulfhydryl or thiol
group.

Acidic or Negatively
charged R groups:
There are two acidic amino acids—aspartic acid and glutamic acid—
whose R-groups contain a carboxyl group. These side chain carboxyl
groups are weaker acids than the -COOH group, but are sufficiently
acidic to exist as –COO- at neutral pH. Aspartic acid and glutamic acid
thus have a net negative charge at pH 7. These negatively charged
amino acids play several important roles in proteins. Many proteins
that bind metal ions for structural or functional purposes possess
metal binding sites containing one or more aspartate and glutamate
side chains.

Basic or Positively charged amino acids:
Three of the common amino acids have side chains with net positive
charges at neutral pH: histidine, arginine, and lysine. The ionized group
of histidine is an imidazol, that of arginine is a guanidinium, and lysine
contains a protonated alkyl amino group. The side chains of the later
two amino acids are fully protonated at pH 7, but histidine is only 10%
protonated at pH 7. Histidine side chains play important roles as proton
donors and acceptors in many enzyme-catalyzed reactions.
Arginine
(Arg, R)

Peptide bond
formation
As both the amine and carboxylic acid groups of amino acids can react
to form amide bonds, one amino acid molecule can react with another
and become joined through an amide linkage. This polymerization of
amino acids is what creates proteins. This condensation reaction yields
the newly formed peptide bond and a molecule of water.

Amino acids can act as Acids and Bases:
Amino acids in aqueous solution are ionized and can act as acids or bases.
When a crystaline amino acid, eg, alanine is dissolved in water, it occurs
as the dipolar ion, which can act either as an acid (proton donar)
H3C-C-COO- H3C-C-COO- + H+
NH3
+
NH2
H H
Or as a base (proton accepter)
H3C-C-COO- + H+ H3C-C-COOH
NH3
+
NH3
+
H H
Substances having
this two-way
property are known
as amphoteric or
ampholytes.

Isoelectric pH of an amino acid: The pH at which an amino acid
is electrically neutral is known as the isoelectric pH for the molecule and
the symbol is pI. The pI value is constant for a particular compound at
specific conditions of ionic strength and temperature.
+ OH-
+ H+
COOH
H3N-CH
CH2
H3C-CH
CH3
+ COO-
H3N-CH
CH2
H3C-CH
CH3
+ + OH-
+ H+
COO-
H2N-CH
CH2
H3C-CH
CH3
Leucine: pI determination
Net Charge: +1,
pH 1.0 (<2.4) Net Charge: 0,
2.4<pH<9.6)
Net Charge: -1,
(pH <9.6)
At pH 1 the ionic form of Leu will have a formal charge of +1. The addition of base
in an amount equal to one half of the moles of leu present in the solution will half
titrate the -COOH of the leu, that is [COO-] / [COOH] =1 and pH=pKa for
COOH=2.4.
Addition of one equivalent of base will completely titrate the -COOH. In this stage
the negatively charged -COO- and positively charged -NH3+ group is equal and the
net charge is zero and this is the zwitterion form of leu.

The zwitterion form is that ionic form in which the positive charge from
positively charge ionized groups is exactly equal to the negative charge
from negatively charge ionized groups of the molecule. Accordingly, the
net charge on a zwitterion molecule is zero, and a zwitterion molecule will
not migrate toward either the cathode or anode in an electric field.
The further addition of 0.5 equiv of base to the zwitterion form of
leucine will half titrate the -+NH3 group. At this point [NH2]/[+NH3]=1
and pH=pKa for +NH3=9.6.
The addition of a further 0.5 equiv of base will completely titrate the
-NH3 group. The pH of the solution is greater than 9.6 and net charge
is -1.
The pI is average of the two pKa values that form the zwitterion form.
For leucine,
pI=(pKa of COOH + pKa of NH3)/2= (2.4+9.6)/2=6.0

Levels of protein structure
There are four distinct levels of protein structure:
1. Primary structure
The sequence of amino acids in a protein is called the primary structure of
the protein. The primary structure is held together by covalent or peptide
bonds, which are made during the process of protein biosynthesis. The two
ends of the polypeptide chain are referred to as the carboxyl terminus (C-
terminus) and the amino terminus (N-terminus).
The primary structure of a protein is determined by the gene corresponding
to the protein. The sequence of a protein is unique to that protein, and
defines the structure and function of the protein. Post-translational
modifications such as disulfide bond formation, phosphorylations and
glycosylations are usually also considered a part of the primary structure,
which cannot be read from the gene.
Understanding the primary structure of protein is important because many
genetic diseases result in proteins with abnormal amino acid sequences,
which causes improper folding and loss or impairment of function. If the
primary structures of normal and mutated proteins are known, this
information may be used to diagnose or study the disease.

2. Secondary structure
The polypeptide backbone of a protein does not assume a random
three dimensional structure, but instead generally forms regular
arrangements of amino acids that are located near to each other
in the linear sequence. These arrangements are termed as
secondary structure of the polypeptide. Secondary structure
refers to highly regular local sub-structures. Two main types of
secondary structure are the alpha helix and the beta strand or
beta pleated sheet.

-helix
Alpha Helix:
In the alpha helix, the polypeptide chain is coiled tightly
in the fashion of a spring. The "backbone" of the
peptide forms the inner part of the coil while the side
chains extend outward from the coil. The helix is
stabilized by hydrogen bonds between the >N-H group
of one amino acid and the >C=O group on the 4th amino
acid away from it.
One "turn" of the coil requires 3.6 amino acid units. The
helix can be either right-handed or left-handed. The
naturally occurring alpha helixes found in proteins are
all right-handed. Not all proteins have a helical
structure, since some do not have it at all and are
random.
Fibrous proteins and keratins are examples of alpha
helix and they are the major component of tissues like
hair and skin.

Some amino acids are not compatible with the -helix
If a polypeptide chain has many glutamic acid residues in a long
block, this segment of the chain will not form an -helix at pH 7.0.
The reason is that the negatively charged COO- groups at adjacent
residues of glutamic acid repel each other so strongly that they
overcome the stabilizing influence of hydrogen bonds on the -helix.
For the same reason if there are many closely adjacent lysine and /
or arginine residues, whose R groups has net positive charge at pH
7.0, they will also repel each other and prevent formation of the -
helix. Serine, threonine and leucine also tend to prevent formation
of the -helix if they occur close together in the chain.

Beta strand/beta pleated sheet:
The beta sheet is another form of secondary
structure in which all of the peptide bond
components are involved in hydrogen bonding.
The surface of beta sheet appear pleated and
these structure is often called beta pleated
sheets.
Silk is an example of the beta pleated sheet.
The intermolecular hydrogen bonding in the
beta-pleated sheet is in contrast to the
intramolecular hydrogen bonding in the alpha-
helix.
The hydrogen on the amide of one protein
chain is hydrogen bonded to the amide oxygen
of the neighboring polypeptide chain. The
pleated sheet effect arises form the fact
that the amide structure is planar while the
"bends" occur at the carbon containing the
side chain.

Antiparallel
Parallel
The beta pleated sheet
can be formed from
two or more separate
polypeptide chains or
segments of
polypeptide chains that
are arranged either
parallel (all N terminals
on one end) or anti-
parallel (N terminal and
C terminal ends
alternate).

3. Tertiary structure
Tertiary structure refers to three-dimensional structure of a
single polypeptide molecule where both to the folding of domains
and to the final arrangement of domains in the polypeptide. The
alpha-helices and beta-sheets are folded into a compact globule.
Hydrophobic side chains are burried in the interior, whereas
hydrophilic groups are generally found on the surface of the
molecule.
Domain: Some polypeptide chains fold into several compact regions.
These regions in a polypeptide chain are called domains and
generally range from 30 to 400 amino acids. On average, domains
contain roughly 100 amino acids. Each domain forms its own tertiary
structure which contributes to the overall tertiary structure of the
protein. These domains are independently stable.

Fig Tertiary structure:
Beta pleated sheats (ribbons with
arrows) and the alpha helical
regions (barrel shaped structures
Forces stabilize the Tertiary
Structure:
• Hydrogen bonding between R groups of
amino acids in adjacent loops of the
chain.
• Ionic attractions between oppositely
charged R groups. As an example the
negatively charged carboxyl group of a
glutamic acid residue may be attracted
to the positively charged epsilon amino
group of a lysine residue in an adjacent
loop.
• Hydrophobic interactions
• Covalent cross linkages: disulfide cross
linkage by cystine amino acid

4. Quaternary structure
Many proteins consists of a single polypeptide chain, and are
defined as monomeric protein. However, others may consist of two
or more polypeptide chains that may be structurally identical or
totally unrelated. The arrangement of these polypeptide subunits
called the quaternary structure of the protein. Subunits are held
together by noncovalent interactions, for example, hydrogen bond,
ionic bond and hydrophobic interactions. Subunits may either
function independently of each other or may work cooperatively,
as in hemoglobin (two alpha and two beta chains), in which the
binding of oxygen to one subunit of the tetramer increases the
affinity of other subunits for oxygen.

Bovine hemoglobin is composed of two pairs of non-identical subunits,
alpha and beta. Each alpha-beta pair is more closely associated than
they are with each other, but the overall arrangement is roughly
tetrahedral.
Quaternary Structure is the
combination of two or more
chains, to form a complete
unit. The interactions between
the chains are not different
from those in tertiary
structure, but are
distinquished only by being
interchain rather than
intrachain.
Fig Quaternary structure: Bovine hemoglobin

Protein Introduction

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Protein Introduction