Download 04. Proteins

AMINO ACIDS
Proteins - biopolymers
constructed from amino acids
connected to each other by the
peptide bonds
“Protein” - from the Greek word
proteios, meaning primary or first rank
Proteins:
• most abundant macromolecules in living
systems;
• occur in great variety, most diverse
macromolecules in living systems;
• function in all biological processes
Amino acids - building blocks of proteins
Amino acid - any organic molecule with at
least one carboxyl group and at least one
amino group
Several hundreds of different amino acids
are known
In animal cells there are 20 main amino acids
that are genetically coded for incorporation
into protein
20 amino acids found in proteins
3 letter abbreviation or 1 letter symbol are used to indicate the
composition and sequence of amino acids in proteins
General formula of amino acid
side chain
Amino acid has a central tetrahedral carbon atom (α-carbon) linked to:
- an amino group
- a carboxylic acid group
- a hydrogen atom
- side chain (R group)
Because of functional groups are placed around the α-carbon
center, they are called α-amino acids
α-Amino acids are chiral - 4 different groups connected to the
tetrahedral α-carbon (except for glycine which is achiral)
Classification of amino acids
The feature making each amino acid chemically
and biologically distinct is the R side chain
20 side chains are varied in:
 size,
 shape,
 charge,
 H-bonding capacity,
 hydrophobic character,
 chemical reactivity
All amino acids are divided into classes based
on polarity of the side chain
Group 1. Amino acids with nonpolar side chain
Glycin, alanin, valine, leucine, isoleucine, proline,
phenylalanine, methyonine, tryptophan
All amino acids of this group have aliphatic or aromatic
groups and therefore have hydrophobic character
Proline
(Pro, P)
Group 2. Amino acids with polar, uncharged side chains
Serine, cysteine, threonine, tyrosine, asparagine,
glutamine
Side chains of these amino acids have heteroatom (N,
O or S) with electron pair available for hydrogen
bonding to water and other molecules
Serine
(Ser, S)
Cysteine
(Cys, C)
Threonine
(Thr, T)
Asparagine
(Asn, N)
Glutamine
(Gln, Q)
Group 3. Amino acids with polar, negatively charged side
chains at physiological pH
Glutamate, aspartate
Contain acidic functional groups
At physiological pH the side chain dissociate protons to
form carboxylate anions
Aspartate
(Asp, D)
Glutamate
(Glu, E)
Group 4. Amino acids with polar, positively charged side
chains at physiological pH
Lysine, arginine, histidine
Contain basic functional groups
Lysine
(Lys, K)
Arginine
(Arg, R)
Structure
of 20
amino
acids in
proteins
Stereochemistry of
Amino Acids
Amino acids exist in two
stereoisomers (enantiomers),
called D- and L-aminoacids
D- and L-isomers are mirror image of
each other
Only the L-isomers are used
as building blocks of proteins
D-isomer
L-isomer
Properties of amino acids
In pure form are wide, crystalline, high-melting solids
Soluble in water and insoluble in organic solvents
Amphoteric properties – depending on medium can act as
acids or basis
With base amino acids react as acids:
With acids amino acids react as bases:
Amino acids are ionized in aqueous solutions
At physiological pH amino acids have a positive and
negative charge on the same molecule (zwitterions)
The net charge of the zwitterion form is 0
Zwitterions – the fully ionized species of amino acids
having one amino group and one carboxyl group
Ionization of amino acids
At high pH, the protonated amino group loses a proton and
amino acid has negative charge
At low pH (acid solution) the dissociated carboxyl group
accept the proton and amino acid has positive charge
PROTEINS
Proteins - biopolymers constructed from amino acids
connected to each other by the peptide bonds
FUNCTIONS OF PROTEINS
 Enzymes (catalysts of biochemical reactions)
All enzymes of living cells are simple or complex proteins
 Structural function
Fibrous proteins collagen and elastin are the constituents of bones, skin,
tendons, cartilage, walls of vessels
Keratin – main component of hair and nails
Proteins of cell membranes
 Immune function
Immunoglobulins (antibodies) are produced in response to the antigen
(bacteria, foreign proteins) invasion into the organism
 Transport function
Hemoglobin – transport of oxygen and carbon dioxide
Lipoproteins – transport of cholesterol, triacylglycerol via the blood
 Storage function
Store nutrients for future use
Casein – protein of milk
Myoglobin – depot of oxygen in muscles
Proteins of blood plasma (albumins and globulins) store
amino acids
 Regulatory function
Many hormones are proteins
 Receptor function
Receptors are located on cell membranes or in the
cytoplasm and play role in the signal generation and
transmission
 Muscle contraction
Actin, myosin, troponin, tropomyosin – functional
components of the contractile system of skeletal
muscles
Levels of
protein
structure
• Primary
• Secondary
• Tertiary
• Quaternary
Primary structure of the proteins
Primary structure - type and sequence of amino
acids in a polypeptide chain
Example: -Met–Glu–Asp–Phe–Gly–Leu–Ala-Met–Glu–Gly–
Amino acid type and sequence is determined
genetically
Primary structure is formed due to the formation of
peptide bonds between amino acids
Peptide bond that is formed
carboxyl group
amino group of
acids
amide bond
between αand αtwo amino
During the formation of
peptide bond the molecule
of H2O is released
Peptide bond is a covalent,
Dipeptide
- two amino
stable bond
acids are linked together
Tripeptide - three amino
acids…
Polypeptides – many amino
acids…
Polypeptides have polarity
Amino and carboxyl groups of amino acids are used for
peptide bond formation
There are free amino and free carboxyl group at the ends of
polypeptide chain
The amino group end is called N-terminal residue and the end
containing free carboxyl group is called C-terminal residue
Polypeptide chain consist of regularly repeating part, so called
backbone, and side chains which are variable in different amino
acids
In the polypeptide chain the carboxyl groups and amino groups are
involved in peptide bond formation and can not dissociate and are
not functionally active
The properties of polypeptide are determined by the side chains of
the amino acid
Structure of the peptide bond
Rotation about the amide
carbon nitrogen (peptide) bond
is limited
Four atoms of the peptide
group are on the same plane
Peptide
bonds are
rigid
Peptide group
is in trans
configuration:
hydrogen atom
of the
nitrogen is
opposite to the
oxygen atom
of the
carbonyl group
Peptide group
acts as single,
planar unit
Naming of peptides
Peptides are named as acyl derivatives (the –ine ending
is changed to –yl) of amino acids listed in the order in
which they appear, starting with the N-terminal amino
acid
Alanyltyrosylglycine
Determination of primary structure of
polypeptides
Sanger’s method
Sanger’s reagent (2,4-dinitrofluorobenzene (DNFB)) reacts with
amino group of the N-terminal amino acid of a polipeptide chain
The carbon-nitrogen
bond between amino
acid and DNFB is
more resistant to
hydrolysis than the
peptide bonds
Substituted
polipeptide is
hydrolized
Terminal amino acid
remains with the
DNFB and can be
isolated and
identified
Edman’s method
Allows to label Nterminal residue and
cleave it without
hydrolyzing other amino
acids
Steps:
1. Phenyl isothiocyanate
react with the amino group
of N-terminal residue
2. Releasing of the modified
amino acid (cyclic derivative)
3. Identifying of the
modified amino acid by
chromatographic procedures
(HPLC)
4. Repeating on next amino
acid….
Secondary structure of protein
molecule
• spatial conformation of the protein backbone
• spatial arrangement of amino acids that are
nearby in sequence
Secondary structure - regularly repeating
conformations of the peptide chain, such as α-helices
and β-sheets
The two most important types of secondary structure:
-α-helix
-β-pleated sheet
α-helix
α-helix - a rodlike structure formed
by tightly coiled polypeptide
backbone
The interaction that hold the polypeptide unit
in the arrangement are hydrogen bonds
between N-H group of one anino acid residue
and C
O group of the amino acid four
residues ahead in the chain
Helix is stabilized by many hydrogen bonds
(which are parallel to long axis of the helix)
Most a helices in
proteins are right
handed (backbone
turns clockwise when
viewed along the axis
from the N terminus)
N
Hydrogen bonds allow
the polypeptide to
stretch up to twice its
normal length
The R-side chains
branch out from
the main chain
Right-handed
(clockwise) rod
O
structure
b-Sheets
In b sheet configuration polypeptide chain form an extended
zigzag
Side chains alternate above and below plane
Multiple polypeptide chains can be arranged side-by-side
Polypeptide chains in b-sheet are stabilized by hydrogen
bonds between C=O and NH on adjacent chains
β-sheet configuration
Tertiary Structure of Proteins
• folding of a polypeptide chain into a closelypacked three-dimensional structure
• the spatial arrangement of amino acids that
are far apart in the sequence (amino acids far
apart in the primary structure may be brought
together)
• stabilized by noncovalent interactions
(e.g. hydrophobic effects, charge-charge
interaction) between side chains or disulfide
bridges
Examples
of tertiary
structure
Quaternary Structure
Monomeric proteins - consist of only one polypeptide
chain
Oligomeric proteins – consist of two or more
polypeptide chain called subunits
Quaternary structure – arrangement and position
of each subunits in the intact protein molecule (3D
structure of 2 or more polypeptide chains)
Subunits are held together by many weak, noncovalent
interactions (hydrophobic, electrostatic)
Example of quaternary structure
Levels of
protein
structure
•Primary
•Secondary
•Tertiary
•Quaternary
Three dimensional conformations of secondary, tertiary and
quaternary structures are held together by a variety of
interactions (bonds) between amino acid side chains
Bonds maintaining spatial conformations of proteins:
-disulfide bonds
-hydrogen bonds
-charge-charge forces (ionic bonds)
-hydrophobic forces
Disulfide bond
Disulfide bond maintains the
secondary, tertiary and
quaternary structures of
proteins
It can be formed between two
cysteines of the same polypeptide
chain or adjacent chains
Disulfide bonds
Formation of disulfide bond
Disulfide
bond – bond
between two
sulfur atom
of amino
acid
cysteine
It is formed
by the
oxidation of
a pair of
cysteine
residues
Hydrogen bonds
Stabilizes secondary, tertiary and
quaternary structures
Hydrogen bonds in secondary structure
occur between N-H group of one anino
acid residue and C
O group of the
amino acid four residues ahead in the
chain
Helix is stabilized by many hydrogen bonds
which are parallel to long axis of the helix
Charge-charge interaction (ionic bonds)
 interaction between two charged amino acids
 negatively charged amino acids: glutamate
and aspartate;
 positively charged amino acids: lysine,
arginine and hystidine
The hydrophobic effect
Nonpolar side chains of amino acids associate with each
other
Nonpolar side chains are in the interior
Nonpolar molecules are aggregated because polar
water molecules push away nonpolar compounds (nonpolar
compounds want to escape from water)
CH3
Loss of Protein Structure
(Denaturation and Hydrolysis)
• Denaturation - disruption of native conformation (loss of
organized structure) of a protein, with loss of biological
activity
• The stabilizing forces holding a protein in its native
conformation are relatively weak so they can be
disrupted under mild conditions
• During denaturation only higher structures (secondary,
tertiary and quaternary) of proteins are disrupted.
Primary structure remains unchanged
• Denaturation is irreversible process
• Some proteins can be refolded (renatured)
Denaturants
• Heat (example: denaturation of egg white)
t > 50oC breaks H-bonds
• Strong acids and alkalines
• Organic solvents (ethyl alcohol, acetone)
• Detergents
The tertiary structure of ribonuclease is maintained by
four S-S bridges
Denaturation of ribonuclease with urea and
mercaptoethanol results in complete loss of tertiary
structure and enzymatic activity and yields a
polipeptide chain containing eight sulfhydryl groups
Hydrolysis – breaking of the peptide bonds with free
amino acid formation
During hydrolysis the primary structure of proteins is
destroyed
Acid and alkaline hydrolysis – boiling of proteins in the
presence of strong acids or bases
Enzymatic hydrolysis takes place in the stomach and
intestine (dietary proteins are hydrolyzed by the
enzymes peptidases)
Examples of proteins and their structure
Fibrous Proteins
• have “fiber-like” or elongated shape
• have highly developed secondary structures
• Keratin is found in hair, feathers, horns (depends on the
–helix for strength)
• Fibroin is folded into b–sheet secondary structure. It is
found in silk
• Collagen is a major protein in connective tissue of
vertebrates (25-35% of total protein in mammals)
- forms the bone matrix, ligaments, tendons and skin
- consists of three helical chains coiled around each other in
supercoil
- is extremely strong and stable
Keratin,
fibroin
and
collagen –
examples
of fibrous
proteins
Globular Proteins
have the structure similar to sphere
• Hemoglobin (Hb)
• tetrameric protein (has quaternary structure)
• carries oxygen in the blood
• transports carbon dioxide from peripheral tissue
to lungs
• maintains the pH of blood (buffer function)
• Myoglobin
• monomeric protein
• present in muscles
• act as a reserve supply of oxygen within a local
region
Hb is an a2b2 tetramer (2 a and 2 b globin subunits)
Each subunit has a heme group
Heme consists of a tetrapyrrole ring system
(protoporphyrin) complexed with iron
Heme of Mb and Hb binds oxygen for transport
(a) Human oxyhemoglobin (b) Tetramer schematic
Heme
Tertiary
structure of
myoglobin
Heme
The structure of
myoglobin is similar to
globin subunit of
hemoglobin
Myoglobin has
greater affinity
for oxygen than
hemoglobin
a-Globin (blue)
b-Globin (purple)
Myoglobin (green)
Immunoglobulins
• Vertebrate immune systems synthesize
protein antibodies (immunoglobulins) to
eliminate bacteria, viruses, other foreign
substances
• Antibodies specifically recognize and bind
antigens (molecules foreign to the body)
• Antibodies are synthesized by lymphocytes
(white blood cells)
• Immunoglobulins are composed of more than
one polypeptide and have quaternary
structure
Diagram of
immunoglobulin
• Heavy chains
(blue) and
light chains
(red)
• Disulfide
bonds
(yellow)
• Variable
domains
colored
darker
Tests for proteins and amino acids
Biuret test
CuSO4 + protein (peptide) + NaOH
At least two peptide bonds must
be present for positive reaction
Amino acids and dipeptides do not
give the positive biuret reaction
Ninhydrin test
Ninhydrin reagent reacts with all
-amino acids (except proline)
with the formation of blue color
violet color
Xanthoproteic test
Proteins containing the benzene ring
(phenylalanine, tryptophan, tyrosine)
react with concentrated nitric acid
to give a yellow reaction product
Lead acetate test
If the protein contains cysteine, the sulfur atom
will react with the lead acetate reagent. Black
precipitate will be formed
Millon’s test
Protein containing tyrosine will give
the positive test (red color) with
Millon’s reagent