Amino Acids and Proteins
Proteins are organic molecules consisting of many amino acids
bonded together.
General Properties
o Most abundant biomolecule; accounts for 50% of dry weight.
o Built by assembling long chains of amino acids (monomers),
followed by intricate folding
o Final shape of protein is very specific. Unless correctly
folded, is not functional.
o several 1000 different types of proteins in any cell;
millions of protein molecules
o To understand cellular life, must understand what different
proteins are doing, how they work. A vast, but doable,
challenge.
Functions
o rigid structure -collagen in connective tissue, bone; keratin
in fingernails and hair; silk fibers
o enzymes ; 3-D stereospecific chemical catalysts accelerate
desired reactions by as much as 10 10 times over their
spontaneous rates..
o transport : membrane transport proteins carry substances
across cell membranes; blood transport proteins that move
certain substances (e.g., iron, oxygen, cholesterol)
throughout the body.
o hormones ; chemical signals. Some hormones consist of as
little as a single amino acid. Others are peptides or
polypeptides. Example: insulin
o contraction ; muscle fibers, cilia, spindle fibers in
mitosis.
o specific binding : e.g., that bind specifically to foreign
substances to identify them to the body's immune system
o buffers; proteins form zwitterions. The amino group accepts a
proton and becomes NH3+ and the carboxyl group (COOH) donates
a proton and becomes dissociated (COO-). The net charge on
protein molecules varies with pH and is zero at the
isoelectric point. Therefore the isoelectric point is
important in considering the behaviour of food proteins
because at this pH many properties are either at maximum or
minimum. For example, electrical conductivity, solubility and
viscosity are all a minimum. Electrical conductivity is
minimum because net charge on each molecule is zero.
Solubility is also minimum because there is a strong
electrostatic attraction between neighbouring molecules hence
molecules pack closely together. Hydrogen bonding also
contribute to the close packing thus reducing the possibility
of interaction with solvent molecules as a result, solubility
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becomes minimum. Insolubility affects gel formation hence
viscosity is also minimum. Amino acids with more amino groups
than COOH are basic, and have isoelectric points above pH 7,
while those with more COOH groups than amino are acid hence
their isoelectric points are below pH 7.
Structure of Amino Acids
o Each amino acid has an amino group and a carboxyl group,
joined by a single Carbon atom. In addition, each amino acid
has a characteristic "side chain" (often called the -R
group). These determine the chemical properties of proteins.
o Each amino acid (except glycine) can occur in two isomeric
forms, L- and D-, because of the possibility of forming two
different enantiomers (stereoisomers) around the central
Carbon atom. Only L-amino acids are found in proteins in all
organisms.
o Note: some D-amino acids are found in bacterial cell walls.
o "R" side group can be any of 20 different chemicals.
o The most common amino acid, glycine, is only mildly nonpolar.
o The great variety of side chains allows proteins to have many
different (chemical) properties, and to create many different
environments.
For example;
a. The presence of hydroxyls, carboxylates, sulfhydryls,
and amino groups allows hydrogen bonding and the alkyl
groups provide hydrophobic interactions, within the
protein polymer itself and between separate protein
molecules.
b. Hydroxyl groups of serine, threonine and hydroxyproline
can participate in ester linkages which have important
influences on the properties of protein, as in the
ability of enzymes to bind cofactors.
c. The phenolic hydroxyl of tyrosine contributes to the
acidic
properties of proteins.
d. Arginine yields a red colour when treated with naphthol and sodium hypochlorite.
Peptides and Polypeptides
a. Amino acids are not accumulated by cells, but quickly
joined into specific assemblies by the formation of
peptide bonds .
b. The peptide bond that binds amino acids is one of the
strongest and most durable of covalent bonds. In the
laboratory, we can break, or hydrolyze, peptide bonds
most effectively by a combination of heat and acid. In
your body, this digestive process begins in the stomach,
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where a combination of acid and enzymes help to break
peptide bonds. If you didn't need to digest proteins,
you wouldn't need a stomach!
c. Two amino acids joined = dipeptide. Three AAs joined =
tripeptide Many AAs joined = polypeptide .
d. Short chains of amino acids with low molecular weight
are called peptides. Polypeptide refers longer chains of
amino acids (aprox. more than 30 amino acids). Every
peptide and polypeptide has one free amino group (called
the "N-terminus") and one free carboxyl group(called the
"C-terminus"). " Protein " refers to the overall
functional assembly, created when one or more
polypeptides fold up and become functional units. Some
proteins consist of only a single folded polypeptide
chain, but many proteins contain multiple polypeptides,
and frequently inorganic atoms as well, such as Zinc,
Iron, Magnesium, etc.
Protein Structure
1. Protein structure is determined by several factors.
These same factors determine the proteins folding, stability,
solubility and purification, function and specificity, etc.
a. Chemical composition
o atoms, bonds, bond types = amino acids
amino acid sequence, peptide bonds = primary sequence
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b. Chemical conformation
side chain conformation
backbone conformation, restricted conformations
alpha helices, beta strands, phi and psi values, etc.
hydrogen bonding
c. Chemical composition and the roles played by each type:
hydrophobic
aromatic
hydrophilic
hydrogen bonding
electrostatic and ionic
2. Amino acids link through peptide bonds to form polymers.
o The polymers are called peptides (less than 30) or proteins
(greater than 30).
o The structures of the proteins are determined by the sequence
of amino acids.
o The possible structures of the backbone of the proteins are
restricted by the peptide bond. The peptide bond is planar
about the C, O, N and NH atoms. Due to the bulk of the side
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chain and other atoms the protein has a restricted
conformation about the phi and psi bonds.
Levels of Structure
1 o Structure
o Primary structure is the sequence of amino acids in the
polypeptide chain. This represents the covalent bonding in
the protein molecule. e.g. Val-Leu-Ser-Glu-Gly-Glu-Trp-GlnLeu-Val- represents the first ten amino acids of myoglobin
(out of 153 total).
o Note: since every polypeptide begins with free amino group,
this is called the N-terminus. The opposite end of the
polypeptide has a free carboxyl group, called the C-terminus.
2 o Structure
1. Polypeptides fold in a series of stages. The first level of
folding is called the secondary (2o) structure. Secondary structure
is the organization of the polypeptide into regular repetitive
patterns over short segments of 5-15 amino acids, for example the
helical segments in the myoglobin structure.
2. The alpha-helix is one of the most common 2o folding patterns
discovered by Pauling and Corey.
o Alpha helix: Hydrogen bonds can form readily between C=O
groups in the backbone and N-H groups four amino acid
residues further along the chain.
o This regular pairing pulls the polypeptide into a helical
shape that resembles a coiled ribbon.
3. Another common folding pattern is called beta-pleated sheet. In
this structure, polypeptides may run parallel to each other and
hydrogen bonds are formed between a carbonyl group of one chain
and the hydrogen of the imino group of another, thus linking the
two-polypeptide chains together. The number of hydrogen bonds
formed far much exceeds those in alpha-helix structure. Therefore
the beta-pleated structure is much more stable.
4. Some protein regions remain in random coil, no regular pattern
of secondary structure.
5. Different proteins have different degrees of alpha helix, beta
sheet, and random coil. Silk is a protein stabilized entirely by
pleated sheet; keratin (in hair) is stabilized entirely by alpha
helix. Most proteins have some or both.
6. The nature of the hydrogen bonding confers stability onto the
heling structure and the elasticity of such a molecule is due to
the fact that hydrogen bonds can stretch much more than covalent
bonds. The secondary structure accounts for the marked elasticity
of some fibrous protein like keratin in wool or hair.
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7. The secondary structure is based on the rotationary angles
assumed about the covalent bond interconnecting the amino acid and
the peptide bond. This rotation occurs between;
a. The N and the -Carbon atom (phi)
b. -Carbon atom and the carbonyl carbon. (psi
3o Structure
Tertiary structure is the overall folding of the whole
polypeptide.
o Polypeptides may continue folding beyond the formation of
secondary structure. It is only with the complete, compact
folding into tertiary (3 o ) structure that they attain their
"native conformation" and become active proteins (as a result
of the creation of active sites).
o Forces that contribute to tertiary folding include:
a. Hydrogen bonds
b. Hydrophobic bonds - interactions between nonpolar
regions.
c. Ionic bond - between adjacent side chains.
d. Sulfhydryl bonds (-S-S- bonds). These are especially
important, because they are covalent bonds and quite
strong compared to H-bonds.
4o Structure
Some proteins are made of multiple polypeptide subunits, which
must be assembled together after each individual polypeptide has
reached its 3o structure.
Examples:
a. Hemoglobin (blood protein involved in oxygen
transport) has four subunits.
b. Pyruvate dehydrogenase (mitochondrial protein
involved in energy metabolism) has 72
subunits.
Quaternary structure is the grouping of several protein molecules
into a single larger entity; the subunits may act cooperatively
with each other to give the grouping special properties not
possessed by the single subunit. Not all proteins have a
quaternary structure, e.g. a single myoglobin molecule functions
by itself, but its close relative, hemoglobin, is a tetramer of
four globin subunits. Each globin is structurally very similar to
myoglobin.
The bonding mechanisms that hold the protein chains together are
generally the same as those involved in the tertiary structure
with the exception of disulphide bonds that are considered
unimportant in maintaining the quaternary structures.
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Conjugated Proteins
Proteins may exist in combination with carbohydrates, lipids,
nucleic acids, metal ions or phosphates. These prosthetic groups
are bound to the protein by linkages other than salt linkages. The
conjugation confers stability to the protein. For example
o Lipoproteins: complexes with lipids
o Glycoproteins: conjugated with heterosaccharides.
o Metalloproteins: complexes with heavy metals
o Nucleoproteins: protein and nucleic acid found in viruses and
ribosomes.
o Phosphoproteins: complexes with inorganic phosphate.
Ionic Properties of Proteins
o Hydrophilic R-groups are generally located on the exterior
of the protein molecule, while the hydrophobic or non-polar
are in the interior.
o Titration experiments show that nearly all of the
potentially ionizable R-groups of the protein are capable of
ionizing.
o A protein with a high content of acidic groups amino acids
(aspartic and glutamic) has a low isoelectric point. If it
has more basic amino acid (arginine, lysine) it has a high
isoelectric point.
o If it binds ions of neutral salts, (magnesium & calcium) a
change in both the isoelectric point and the titration curve
occurs.
o Variations in ionic properties pave way for several methods
for fractionation proteins from biological systems to
include, electrophoresis, ion-exchange chromatography.
o Electrical charge also influences solubility. As the
isoelectric point is approached the charge difference
between molecules is lessened, hence proteins touch,
aggregate and sometimes precipitate.
o Proteins that are water-soluble owe their solubility to
water protein hydrogen bonding and the environmental pH that
do not coincide with their electric points. This background
is used to precipitate milk casein. Ions with neutral salts
react with charges on proteins decreasing the electrostatic
attraction between opposite charges of neighboring protein
molecules and increase in solvation and enhance their
solubility. This is called 'salting in'. Precipitation
occurs with an increase in the concentration of neutral
salts - 'salting out'.
o Organic solvents eg. acetone, ethanol, compete with the
protein for water thus decrease the dielectric constant of
the solution. This decreases the repulsive forces of like
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charges making the proteins aggregate and precipitate. This
method can be used to fractionate protein from biological
material.
o Generally, solubility increases with temperatures between
0oC and 40oC except for (- casein which is soluble at 0oC.
Above 40oC proteins denature thus lose solubility.
Denaturation & Renaturation
o When proteins are heated, or exposed to acids or bases, or
high salt concentrations, the variety of weak bonds holding
tertiary and quaternary structure together can be disrupted
so that the protein unfolds. Unfolding = denaturation
resulting in loss of function. Unfolding can proceed even to
disrupt secondary structure. Denaturation is therefore any
modification of the secondary, tertiary or quaternary
structure of a protein molecule, excluding, breakdown of
covalent bonds (Fennema, O, 1985).
o Denaturation is sometimes reversible; an unfolded protein can
be restored to correct folding and regain biological
activity. This is called renaturation, regeneration,
reactivation.
o Denaturation can also occur irreversibly (as when egg white
protein, albumin, is denatured by boiling to congeal as egg
white). Renaturation is then no longer possible.
a. When denaturation occurs, peptide bonds of the protein
are more readily available for hydrolysis by proteolytic
enzymes, solubility is decreased, enzymatic activity is
decreased or lost, intrinsic viscosity is increased and
optical rotation of a solution is increased. Protein
molecules unfold and become more asymmetrical exposing
more hydrophobic residues, thus decreasing solubility
and increasing viscosity.
b. Sensitivity to denaturation agents varies with proteins.
c. Denaturation results differ with conditions and agents
even if the protein molecule is the same.
Denaturation can be brought about by;
a. Controlling pH - This occurs most readily in the
isoelectric point when the proteins are least stable eg.
Caseinogens in milk.
b. Heat causes protein to coagulate. Slowly coagulated
protein is easily digested compared to rapidly done,
which results in a hard solid mass.
Protein coagulation is widely used to recover recombinant
protein products, enzymes and in the production of common
foods. Denaturation by heat treatment is affected by addition
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of mineral salts, which cause the protein to precipitate out
of solution resulting in an insoluble salt.
c. Beating egg white brings about partial coagulation of
ovalbumin resulting in a foam which can be further
coagulated into a rigid structure by heating. Foaming
occurs at isoelectric point when the egg is lest stable.
Addition of an acid improves foaming.
Effects of denaturation
o Peptide bonds become more readily available for
hydrolysis by proteolytic enzymes
o Solubility is decreased-the protein becomes more
assymetrical exposing hydrogen groups hence the
insolubility.
o Enzyme activity is decreased or lost
o Crystalisation is no longer possible
o Intrinsic viscosity increased- structure becomes more
assymetrical.
o Optical rotation of the protein is increased.
Gelation - Like carbohydrates, proteins are used to form gels.
Proteins are able to bind by hydration approx. 1g of water per 5g
of dry protein. Some proteins form gels by immobilizing water
within the network. This water is physically entrapped and can be
expelled from the gel when stress is applied. Immobilized water
does not flow freely from a gel. Proteins with a high degree of
asymmetry are capable of forming 3 dimensional matrix by
establishment of interprotein hydrogen bonding. This cross-linking
makes it possible for the structure to hold water in an
immobilized state. The gel shrinks when pH is changed to approach
isoelectric point, expelling some of the immobilized water through
synerisis.
Food Uses of protein
o Proteins are used for their thickening, gelling, emulsifying
and water binding properties in meats (sausages), bakery
products, cheese, deserts and salad dressings.
o They are used for their cohesive and adhesive properties in
sausage making, pasta and baked goods.
o Egg proteins are used for their foaming properties in
desserts, cakes and whipped toppings.
o Milk, egg and cereal proteins are used as fat and flavour
binders in low fat bakery products.
o Proteins are used for texture and palatability in bakery
products
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