Chapter 1

Protein structure and function

Cai Wangwei, Professor, Ph D

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E-mail: [email protected]
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 Learning objectives: students will be able to
1. Know the common features of amino acids and T the
classification of amino acids.
2. Know the structure of peptide bond, the structure of peptide
unit and peptide chain.
3. Know the primary, secondary, tertiary and quaternary structures
of protein.
4. Know the structure and function of myoglobin and hemoglobin.
5. Understand the physicochemical properties of protein and their
applications.
 Overview
Proteins are the most abundant and functionally diverse molecules in
living systems.
Proteins account for 50% of the dry weight of the human body.
Every life process depends on protein molecules. Proteins are working
molecules in all organisms
For example,
enzymes and polypeptide hormones : direct and regulate metabolism in the
body
contractile proteins in muscle : movement.
In bone:
 Collagen forms a framework for the deposition of calcium
phosphate crystals, acting like the steel cables in reinforced
concrete.
In the bloodstream:
Hemoglobin and plasma albumin: shuttle molecules essential to
life.
 Immunoglobulins: fight infectious bacteria and viruses.
 About 100,000 different proteins in human body.

 Unlike lipids and carbohydrates, proteins are not

stored, so they must be consumed daily.

 Current recommended daily intake for adults is

0.8 grams of protein per kg of body weight (more
is needed for children).
Biological Function of Protein?
 Classification of Proteins by Biological
Function
 Enzymes (lactate dehydrogenase, DNA polymerase)

 Storage proteins (ferritin, cassein, ovalbumin)

 Transport proteins (hemoglobin, myoglobin, serum

albumin)

 Contractile proteins (myosin, actin)

 Hormones (insulin, growth hormone)

 Protective proteins in blood (antibodies, complement,

fibrinogen)

 Structural proteins (collagen, elastin, glycoproteins)

Proteins are the working molecules in all organisms

 Section 1
Structure of the amino acids

Amino acids that described in nature are more than 300 different
amino acids BUT, only 20 are constituents of mammalian proteins.

These 20 A.A. are the only A.A. coded by DNA (the genetic
material in the cell).
To understand protein function, we must first understand
the nature of amino acids.
Every amino acid has the same basic structure. Each amino acid (except
proline) has a carboxyl group, an amino group, and a distinctive side chain
“R-group” bonded to the -carbon atom
Carboxylic acids are traditional Bronsted-Lowery acids, donating a
proton in aqueous solution.
R-COOH  R-COO- + H+
pH= [less than 2]  [above 5]

Amino groups function as bases, accepting a proton.

R-NH3+  R-NH2 + H+
pH= [below 8]  [above 9]

10
Even though both acids and amines are present in the same molecule,
they mostly behave as they are separate entities:

11
At physiologic pH (pH 7.4), the carboxyl group is dissociated to form the negatively
charged carboxylate ion (─COO- ), and the amino group is protonated (─NH3+).

Neutral molecule with equal number of + and – charges is a zwitterion

 In proteins, the carboxyl and amino groups are combined in peptide
linkage and, available only for hydrogen bond formation.

Amino acids joined by dehydration synthesis peptide bond

 Each has a unique side chain, called an R group. The specific
properties of amino acids depend on the nature of their R groups
 The side chains ultimately dictate the role an amino acid plays in a
protein
Amino acids are therefore classified according to the
properties of their side chains :
Hydrophobic: Gly, Ala, Val, Leu, Ile, Phe, Pro.

Polar, uncharged: Trp, Ser, Tyr, Cys, Met, Asn, Gln, Thr.

Acidic: Asp, Glu.

Basic: Arg, Lys, His.

A. Hydrophobic amino acids(amino acids with nonpolar side chains:

Each of these A.A. has a nonpolar

side chain that does not bind or

give off protons or participate in

hydrogen or ionic bonds.

 The side chains of the nonpolar
amino acids can be thought of as
"oily" or lipid-like, a property 
promotion of hydrophobic
interactions.
 Proline:
The side chain of proline and its
 -amino group form a ring
structure, and thus proline differs
from other amino acids in that it
contains an imino group, rather than
 The unique geometry of proline
an amino group. contributes to the formation of the
fibrous structure at collagen, and
often interrupts the -helices found
in globular proteins.
B. Polar uncharged amino acids( with uncharged polar side chains)

• These A.A. have zero net charge at neutral pH. Although the side chains
of cysteine and tyrosine can lose a proton at an alkaline pH
 Serine, threonine and tyrosine each
contain a polar hydroxyl group that can
participate in hydrogen bond formation.

 The side chains of asparagine and

glutamine each contain a carbonyl group
and an amide group, both of which can
also participate in hydrogen bonds.
C. Acidic amino acids(with acidic side chains)
The amino acids aspartic and glutamic acid are proton donors.

At neutral pH, the side chains of these amino acids are fully ionized, containing a
negatively charged carboxylate group (─ COO-). They are, therefore, called aspartate or
glutamate.

glutamic acid
D. Basic amino acids(with basic side chains)

The side chains of the basic A.A. accept protons.

At physiologic pH:

The side chains of lysine and arginine are fully ionized

and positively charged.

In contrast:

Histidine is weakly basic, and the free amino acid is

largely uncharged at physiologic pH.
 20 Standard Amino Acids
Name 3-letter 1-letter Name 3-letter 1-letter
Alanine Ala A Leucine Leu L
Arginine Arg R Lysine Lys K
Asparagine Asn N Methionine Met M
Aspartate Asp D Phenylalanine Phe F
Cysteine Cys C Proline Pro P
Glutamine Gln Q Serine Ser S
Glutamate Glu E Threonine Thr T
Glycine Gly G Tryptophan Trp W
Histidine His H Tyrosine Tyr Y
Isoleucine Ile I Valine Val V
F. Optical properties of amino acids

 The -carbon of each amino acid is attached to four

different chemical groups: a chiral or optically active
carbon atom.

 Glycine is the exception because its -carbon has two

hydrogen substituents, optically inactive.
Amino acids that have an asymmetric center at the -
carbon can exist in two forms, designated D and L, that
are mirror images of each other.
The two forms in each pair are termed stereoisomers,
optical isomers, or enantiomers.
Plane of symmetry

 All amino acids found in proteins are of L- form

 However, D-amino acids are found in some antibiotics and
in bacterial cell walls.
G. Physicochemical Properties of Amino Acids

1) Isoelectric point: at physiological pH, amino acids exist as

dipolar ion (zwitterion)
R-CH-COOH
NH2

+OH- +OH-
R-CH-COOH R-CH-COO- R-CH-COO-
+H+ +H+
NH3+ NH3 + NH2
cation zwitterion anion
Therefore, the isoelectric point (pI) of an amino acid is the pH
at which it has no net charge.

When pH=pI, net charge=0(zwitterion);

When pH>pI, net charge<0(negative);
When pH<pI, net charge>0(positive).
Roughly calculate the pI for an amino acid:

A) If:
K1 K2
+H +H - H2N-R-COO-
3N-R-COOH 3 N-R-COO

then: pI= (pK1+ pK2)/2

B) If:
K1 K2 K3
+H N-R-COOH +H N-R-COO- +H N-R-COO- H2N-R-COO-
3 3 3

COOH COOH COO- COO-

then: pI= (pK1+ pK2)/2

C) If:
K1 K2 K3
+H N-R-COOH +H N-R-COO- H2N-R-COO- H2N-R-COO-
3 3

NH3+ NH3+ NH3+ NH2

then: pI= (pK2+ pK3)/2

2)Ultraviolet light absorption: some amino acids such as Trp and Tyr have
a peak absorption at a wavelength of 280nm.
This property is commonly used to determine the concentration of
proteins.
3) Ninhydrin reaction:

Ninhydrin + amino acid blue-purple compound

This reaction is also used to quantitatively determine amino acids

and proteins.
Section II
Peptide
 A. Peptide bond

• In proteins, amino acids are joined covalently by peptide bonds,

( which are amide linkages between the -carboxyl group of
one amino acid and the -amino group of another).
 For example: valine and alanine, form the dipeptide valylalanine
through the formation of a peptide bond.
 Characteristics of the peptide bond:
 The peptide bond has a partial double-bond character, that is, it is
shorter than a single bond, and is rigid and planar due to resonance that
limits rotation about this bond(The carbonyl oxygen has a partial
negative charge and the amide nitrogen has a partial positive charge,
setting up a small electric dipole)
 The partial double-bond character prevents free rotation around the bond
between the carbonyl carbon and the nitrogen of the peptide bond.
 However, the bonds between the -
carbons and the -amino or -carboxyl
groups can be freely rotated (although
they are limited by the size and
character of the R-groups).

 This allows the polypeptide chain to

assume a variety of possible
configurations.
 The peptide bond is generally a trans bond (instead of cis) in large part
because of steric interference of the R-groups when in the cis position.
 Peptide bonds are not broken by conditions that denature proteins
(e.g. heating or high concentrations of urea).

 Nonenzymic hydrolysis of these bonds requires prolonged

exposure to a strong acid or base at high temperatures .
B. Peptide unit
 A rigid and planar structure made up of four atoms (CO-NH) around
the peptide bond.

 Because of the partial double bond character of the peptide bond,

the peptide unit is always maintained in a trans form.
Amide Planes
C. Peptide

 A linear structure formed by two or more amino acids linked via peptide bonds.
e.g. dipeptide, tripeptide, and octapeptide, are formed from two, three, and eight
amino acids, respectively.

Generally, a peptide containing 2-10 amino acids is called oligopeptide; more

than 10 amino acids form a longer peptide called polypeptide.

Amino acids in a peptide are called amino acid residues.

Human Insulin
D. Important peptides in the body
There are some biologically active peptides in the body. They play important
roles in metabolism or in cell functioning.

1)Glutathion (GSH): formed from Glu,Cys, Gly.

H2N-CH-CH2-CH2-CO-NH-CH-CO-NH-CH2-COOH

COOH CH2

SH
Glu Cys Gly
GSH is a potent reductant that protects macromolecules from
oxidation by H2O2.

H2O2 2GSH NADP +

2H2O GS-SG NADPH + H+

b) Peptide hormones:
e.g. adrenocorticotropic hormone (ACTH, a 39-peptide),
thyrotropin releasing hormone (TRH, a tripeptide).

c) Neuropeptides: in nervous system.

e.g. enkephalin (a pentapeptide).
 Section III
The structure of protein
 The 20 amino acids commonly found in proteins are joined together by
peptide bonds.
 The linear sequence of the linked amino acids contains the information
necessary to generate a protein molecule with a unique three-dimensional
shape.
 The complexity of protein structure is best analyzed by considering the
molecule in terms of four organizational levels, namely: primary,
secondary, tertiary, and quaternary .
 Linkage of many amino acids through peptide bonds to form an
unbranched chain called a polypeptide---- copolymer of amino acids.

Definition:
Amino acid polymers of ≤50 amino acids are called “polypeptides,
peptides, oligopeptides, etc.”
Amino acids polymer of >50 amino acids are called “proteins.”
 Primary

The four levels of protein Secondary

structure

Tertiary

Quaternary

Figure 2.1
 Understanding the primary structure of proteins is important
because many genetic diseases result in proteins with
abnormal amino acid sequences, improper folding and loss or
impairment of normal function.

 If the primary structures of the normal and the mutated

proteins are known, diagnosis or studying the disease.
 A. Primary structure of proteins

 The sequence of amino acids in a protein is called the primary structure of the
protein.
 Linked by peptide bonds (The force to maintain the primary structure)
 Linear sequence

In some cases disulfide bond and covalent cross-link are also involved.
----CH---- ----CH----
CH2 CH2
SH Oxidation S

SH Reduction S
CH2 CH2
----CH---- ----CH----

Formation of a disulfide bond

 B. Secondary structure of proteins
The polypeptide backbone does not assume a random three-
dimensional structure, but instead generally forms the regular
folding regions of the polypeptide chain that are located near to
each other in the linear sequence.
These arrangements are termed the secondary structure of the
polypeptide.
The force to maintain the secondary structure of a protein is
hydrogen bond.
The -helix, -sheet, and -bend are examples of secondary
structures frequently encountered in proteins.
The irregular secondary structures are termed random coil or loop
conformation.
Myoglobin structure
 1. -Helix
-helix is the most common polypeptide
helices found in protein

It is a spiral structure, consisting of a

tightly packed, coiled polypeptide
backbone core, with the side chains of the
component amino acids extending
outward from the central axis to avoid
interfering sterically with each other.
 (1) Hydrogen bonds:
An -helix is stabilized by extensive hydrogen
bonding between the peptide-bond carbonyl
oxygens and amide hydrogens that are part of
the polypeptide backbone.

 The hydrogen bonds extend up the spiral from

the carbonyl oxygen of one peptide bond to the
-NH- group of a peptide linkage four residues
ahead in the polypeptide(Carboxyl group of
residue i hydrogen bonds to amino group of
residue i+4)
This ensures that all but the first and last
peptide bond components are linked to
each other through hydrogen bonds.

Hydrogen bonds are individually weak,

but they collectively serve to stabilize
the helix.
 (2) Amino acids per turn:
Each turn of an -helix contains 3.6 amino acid residues.
Thus, amino acid residues spaced three or four apart in the primary
sequence are spatially close together when folded in the -helix.
5.4 angstroms(0.54nm) in length per turn
A very diverse group of proteins contains -
helices. For example, the keratins are a family
of closely related, fibrous proteins whose
structure is nearly entirely -helical.

They are a major component of tissues such as

hair and skin, and their rigidity is determined
by the number of disulfide bonds between the
constituent polypeptide chains.

In contrast to keratin, myoglobin, whose

structure is approximately eighty percent -
helical, is a globular, flexible molecule.
 (3) Amino acids that disrupt an -helix:
Proline disrupts an -helix because its imino group is not geometrically
compatible with the right-handed spiral of the -helix.
Instead, it inserts a kink in the chain, which interferes with the smooth,
helical structure.
Large number of charged amino acids (glutamate, aspartate, histidine,
lysine, or arginine) disrupt the helix by forming ionic bonds, or by
electrostatically repelling each other.

glutamic acid
Amino acids with bulky side chains, such as tryptophan, or
amino acids, such as valine or isoleucine, ( that branch at the -carbon,
the first carbon in the R-group, next to the -carbon) can interfere with
formation of the -helix if they are present in large numbers.
 2. -Sheet
The -sheet is another form of secondary structure: extended structure.

The surfaces of -sheets appear "pleated" and these structures are, therefore, often called
“ - pleated sheets".

The majority of β-strands are arranged adjacent to other strands.

 Forms parallel and antiparallel pleated sheets

 Hydrogen bond between groups across strands

 Residues alternate above and below β-sheet

When illustrations are made of protein structure, -strands are
often visualized as broad arrows.
 When viewed edge-on, form a zigzag or pleated pattern in which the R groups of adjacent
residues point in opposite directions

 The peptide backbone of the β sheet is highly extended

  -sheets stability is also maintained by hydrogen bonds between the carbonyl oxygens
and amide hydrogens of peptide bonds. These bonds are formed with adjacent segments
of β sheet
 Parallel and antiparallel sheets:

A -sheet can be formed from two or more

separate polypeptide chains or segments
of polypeptide chains that are arranged
either antiparallel to each other (with the
N-terminal and C-terminal ends of the -
strands alternating ), or parallel (with all
the N-termini of the -strands together).
When the hydrogen bonds are formed between the polypeptide
backbones of separate polypeptide chains, they are termed
interchain bonds.

A -sheet can also be formed by a single polypeptide chain

folding back on itself . In this case, the hydrogen bonds are
intrachain bonds.
 3. Comparison of a -sheet and an -helix:

 Unlike the -helix, -sheets are composed of two or more peptide

chains (-strands), or segments of polypeptide chains, which are
almost fully extended.
 in -sheets the hydrogen bonds are perpendicular
to the polypeptide backbone.
 4. -Bends (reverse turns)

-Bends reverse the direction of a polypeptide chain, forming a compact,

globular shape.

They are usually found on the surface of protein molecules, and often include
charged residues.

-Bends were given this name because they often connect successive strands
of antiparallel -sheets.
-Bends are generally composed of four amino acids, one of which may be
proline — the imino acid that causes a "kink" in the polypeptide chain.

Glycine, the amino acid with the smallest R-group, is also frequently found in -
bends.

- Bends are stabilized by the formation of hydrogen and ionic bonds.
 5. Nonrepetitive secondary structure
Approximately, one half of an average globular protein is organized into repetitive
structures, such as the -helix and/or -sheet.
The remainder of the polypeptide chain is described as having a loop or coil
conformation.
These nonrepetitive secondary structures are not "random", but simply have a less
regular structure than those described above.
 Motif
 Motif, also called a fold or (more rarely) supersecondary structure.

 A motif or fold is a recognizable folding pattern involving two or more elements of

secondary structure and the connection(s) between them.

 A motif can be very simple, such as two elements of secondary structure folded
against each other, and represent only a small part of a protein.

 Protein motifs are the basis for protein structural classification

Examples include the , the
hairpin loop, and the helix-turn-helix
motifs
 C. Tertiary structure of globular proteins

The primary structure of a polypeptide chain determines its tertiary structure. The
tertiary structure reflects the unique aspect of the amino acid sequence because it
depends on interactions of the R groups
refers to the three-dimensional arrangement of the atoms of the protein molecule
 Tertiary structure refers to the folded 3D
structure of a protein. It is also known as
the native structure or active conformation.
 "Tertiary" refers both to the folding of
domains (the basic units of structure and
function), and the final arrangement of
domains in the polypeptide.
 The structure of globular proteins in aqueous solution is compact, with a high-
density (close packing) of the atoms in the core of the molecule.

Tertiary Structure the Protein Myoglobin

 It results from the sum of hydrophobic
residues avoiding water, hydrophilic
residues interacting with water, the
repulsion of similarly charged residues, and
attraction between oppositely charged
residues.

 Hydrophobic side chains are buried in the

interior, whereas hydrophilic groups are
generally found on the surface of the
molecule.
All hydrophilic groups (including components of the peptide bond) located in the
interior of the polypeptide are involved in hydrogen bonds or electrostatic
interactions.
 Interactions stabilizing tertiary structure
The unique three-dimensional structure of each polypeptide is determined by its
amino acid sequence.

 Interactions between the amino acid side chains guide the folding of the
polypeptide to form a compact structure.

Tertiary structure mostly is stabilized by noncovalent interactions between

secondary structure elements and other internal sequence regions that cannot be
classified as a particular type of secondary structure.

Four types of interactions cooperate in stabilizing the tertiary structures of globular

proteins.
Interactions Involved in Tertiary Structure

Hydrogen
bond
Hydrophobic
interactions and
van der Waals
interactions
Disulfide
bridge
Ionic bond

Polypeptide
backbone
 Disulfide bonds:

A disulfide bond is a “ covalent

linkage formed from the sulfhydryl
group (- SH) of each of two cysteine
residues, to produce a cystine
residue” .
 The two cysteines may be separated
from each other by many amino acids
in the primary sequence of a
polypeptide, or may even be located on
two different polypeptide chains; the
folding of the polypeptide chain (s)
brings the cysteine residues into
proximity, covalent bonding of their
side chains.
The primary structure of insulin:

 Is a hormone that regulates the glucose level

in the blood.

 Chain A has 21 amino acids and chain B has

30 amino acids.

 Contains of two polypeptide chains linked by

disulfide bonds (formed by side chains (R)).
Immunoglobulin
A disulfide bond contributes to the
stability of the three-dimensional shape
of the protein molecule.

For example, four disulfide bonds are

found in proteins such as ribonuclease.

These strong, covalent bonds to

stabilize the structure of proteins, and
prevent them from becoming denatured
in the extracellular environment.
 Hydrophobic interactions:

Amino acids with nonpolar side chains

tend to be located in the interior of the
polypeptide molecule, where they
associate with other hydrophobic amino
acids.
 The amino acids with polar or charged side chains tend to be located
on the surface of the molecule in contact with the polar solvent.
 3. Hydrogen bonds:

Amino acid side chains containing oxygen - or nitrogen - bound

hydrogen, such as in the hydroxyl groups of serine and threonine, form
hydrogen bonds with electron-rich atoms, such as the oxygen of a
carboxyl group or carbonyl group of a peptide bond.
Formation of hydrogen bonds between polar groups on the surface of
proteins and the aqueous solvent to enhances the solubility of the protein.
 4. Ionic interactions:

Negatively charged groups, such as the

carboxyl group(-COO-) in the side chain
of aspartate or glutamate, can interact
with positively charged groups, such as
the amino group(-NH3+ ) in the side
chain of lysine.
Interactions Involved in Tertiary Structure
 Domain.

 A domain is a discrete locally folded unit of tertiary

structure, usually with a specific function

 A domain, is a part of a polypeptide chain that is

independently stable or could undergo movements as
a single entity with respect to the entire protein.

 A domain is typically 50-350 amino acids long, with

regions of  helices and  sheets packed together

 Proteins with similar functions often share a common

domain
 V. Quaternary structure of proteins

Many proteins consist of a single polypeptide chain, and are defined as

monomeric proteins.

However, others may consist of two or more polypeptide chains that

may be structurally identical or totally unrelated.

The arrangement of these polypeptide subunits is called the quaternary

structure of the protein.
Hemoglobin is a protein tetramer, containing two identical pairs
of subunits:

22
• Two subunits, the protein is “dimeric”.
• Three subunits “trimeric”.
• Several subunits. ‘multimeric’

• Subunits are held together by noncovalent interactions (e.g., hydrogen

bonds, ionic bonds and hydrophobic interactions).
Subunits may either
function independently of each other, or work cooperatively,

 As in hemoglobin, in which the binding of oxygen to one subunit of

the tetramer increases the affinity of the other subunits for oxygen.
 Section IV
Hemeproteins
Hemeproteins are a group of specialized proteins that contain heme
as a tightly bound prosthetic group.
Heme
Myoglobin
Hemoglobin
Some protein in respiratory chain
Cytochrome
Catalase
A. Structure of heme
Heme is a complex of protoporphyrin IX and ferrous iron (Fe2+)

The iron is held in the center of

the heme molecule
Iron can form six bonds:
four with porphyrin nitrogens
two additional bonds:one
above and one below the planar
porphyrin ring
In myoglobin and hemoglobin, one of these
positions is coordinated to the side chain of a
histidine residue (the nitrogen of a histidine
imidazole, known as proximal His) of the globin
molecule, whereas the other position is available to
bind oxygen.
 B. Structure and function of myoglobin
Myoglobin is a hemeprotein present in heart and skeletal muscle
Myoglobin is storage of O2 in muscles. During periods of oxygen deprivation,
oxymyoglobin releases its bound oxygen.
 Structure of myoglobin

 Myoglobin is a monomeric protein that is mainly

found in in muscle tissue.
 It consists of a single polypeptide chain of 153
amino acid residues and a heme prosthetic group
 It includes a prosthetic group, the heme group
 It can be present in two forms:
 oxymyoglobin (oxygen-bound)
 deoxymyoglobin (oxygen-free)

Mb + O2 MbO2
 1. -Helical content:

The tertiary structure of myoglobin:

Compact molecule
 it has a compact structure with 80% of
the polypeptide chain in 8 -helices,
referred to as “A” through “H”, that are
connected by short non-helical regions.
 2. Location of polar and nonpolar amino acid residues:

 Like other globular protein, amino acid

R-groups exposed on the surface of the
molecule are generally hydrophilic,
while those in the interior are
predominantly hydrophobic.
 Except for two histidine residues in
helices E and F (known as E7 and F8)
F8 His is designated as proximal His,
whereas E7 His is known as distal His
 3. Binding of the heme group:
The heme group of myoglobin sits in a crevice
in the molecule, which is lined with nonpolar
amino acids.
The proximal histidine (F8), binds directly to
the iron of heme. The second, or distal histidine
(E7), does not directly interact with the heme
group, but helps stabilize the binding of oxygen
to the ferrous iron.
The conformation change of myoglobin
creates a special microenvironment for the heme
that permits the reversible binding of one oxygen
molecule
C. Structure and function of hemoglobin
Hemoglobin:
 transport of O2 and CO2
 It binds oxygen efficiently and becomes
saturated at the high oxygen pressure found
in lungs (approximately 100 mm Hg)
 It releases oxygen and become unsaturated
in tissues where the oxygen pressure is low
(about 30 mm Hg)
 It can transport H+ and CO2 from the tissues
to the lungs

Hb + 4O2 Hb(O2)4

 blood buffering
1.Quaternary structure of hemoglobin:

Hemoglobin:
Hemoglobin is tetrameric
hemeprotein (four protein chains HbA(22)
known as globins with each bound HbA2 (22)
to heme. HbF (22) :Fetal hemoglobin
In adults, the four globin proteins
are of two different types known
as α and β, so a hemoglobin
protein is an α2β2 globin protein.
The α and β chains contain
multiple α-helices where α
contains 7 α-helices and β contains
8 α-helices (similar to myoglobin).
 The hemoglobin tetramer can be envisioned as being composed of two identical
dimers,(αβ)1 and (αβ)2, in which the numbers refer to dimers one and two.
 The two polypeptide chains within each dimer are held tightly together, primarily
by hydrophobic interactions .
 The forces to maintain the two dimers ((αβ)1 and ,(αβ)2) include ionic and
hydrogen bonds between the members of the dimer.

The location of the ionic bonds

between the αβ dimers.
 The two dimers are able to move with respect to each other, being held together
primarily by polar bonds.
 The weaker interactions between these mobile dimers result in the two dimers
occupying different relative positions in deoxyhemoglobin as compared with
oxyhemoglobin.
Hemoglobin is an allosteric protein.
An allosteric protein: a protein where binding of a molecule (ligand) to one part of
the protein affects binding of a similar or a different ligand to another part of the
protein.
Hemoglobin exists in two forms, T-state and R-state
The T-state is also known as the "taut" or "tense" state and it has a low-binding affinity
to oxygen. The two αβ dimers interact through a network of ionic bonds and hydrogen
bonds that constrain the movement of the polypeptide chains.
The R-state is known as the "relaxed" state and it has 300 times higher affinity to
oxygen than as the T conformation .
Binding of O2 causes the rupture of some of the ionic bonds and hydrogen bonds
between the αβ dimers, resulting in conformational changes in hemoglobin, converting it
from the low affinity T-state to the high affinity R-state . The binding of oxygen to
hemoglobin
When heme is free of oxygen, it has a domed structure and iron is outside the
plane of the heme group.
When oxygen binds to an iron atom, heme adopts a planar structure and the iron
moves into the plane of the heme pulling proximal histidine (F8) along with it.
Binding of O2 causes conformational changes in hemoglobin, converting it from
the low affinity T-state to the high affinity R-state .

Note: The binding of O2 to the heme iron pulls the iron into the plane of the heme. Because the iron is also
linked to the proximal histidine (F8), there is movement of the globin chains that alters the interface between the
αβ dimers.
D. Binding of oxygen to myoglobin and hemoglobin

 Myoglobin can bind only one molecule of oxygen,

because it contains only one heme group.
 Hemoglobin can bind four oxygen molecules.
 The degree of saturation (Y) of these oxygen-binding
sites on all myoglobin or hemoglobin molecules can
vary between zero (all sites are empty) and 100%
The partial pressure of oxygen needed to achieve
half-saturation of the binding sites (p50) is
approximately 1 mm Hg(mercury) for myoglobin and
26 mm Hg for hemoglobin.

The saturation curve of hemoglobin binding to O2 has

a sigmoidal shape(two or more molecules of oxygen
binding), while the saturation curve of myoglobin
binding to O2 has a hyperbolic shape(a single
molecule of oxygen binding).
 Binding of oxygen to myoglobin:

mitochondria
Buffering of O2 : MbO2 Mb + O2

blood
At 100 mm Hg, hemoglobin is 95-98% saturated
(oxyhemoglobin).
As the oxygen pressure falls, oxygen is released to
the cells.
The subsequent binding of oxygen occurs with
high affinity, as shown by the steep upward curve in
the region near 20–30 mm Hg.
 E. Allosteric effects
Hb is an allosteric protein: binding of O2 to one subunit induces conformational change of
other subunits, making them more easy to bind O2.

The ability of hemoglobin to reversibly bind oxygen is affected by

pO2 .

pH.

pCO2.

2,3-bisphosphoglycerate(2,3-BPG).

Allosteric effector: pO2 , pH, pCO2, 2,3-BPG.

Sequential model for the cooperative
transition of Hb subunits

Some oxygen bound. Each binding

High affinity of an oxygen molecule favors the
Transition state transition of adjacent subunits to
state the strong-binding state and
promotes their binding of oxygen
Peripheral
tissues

More oxygen bound. More and

more subunits next to oxygen-
Low affinity
state Lungs occupied sites are switching to
the strong-binding state.
Change in conformation of subunits in tetramer as Hb goes from T -> R state
1. Heme-heme interactions:

 The sigmoidal oxygen dissociation curve reflects

specific structural changes that are initiated at one
heme group and transmitted to other heme groups in
the hemoglobin tetramer.
 The net effect is that the affinity of hemoglobin for
the last oxygen bound is approximately 300 times
greater than its affinity for the first oxygen bound.
a. Loading and unloading oxygen:

The cooperative binding of oxygen

allows hemoglobin to deliver more
oxygen to the tissues in response to
relatively small changes in the partial
pressure of oxygen.
b. Significance of the sigmoidal oxygen dissociation curve:

The steep slope of the oxygen dissociation curve over

the range of oxygen concentrations that occur between
the lungs and the tissues permits hemoglobin to carry
and deliver oxygen efficiently from sites of high to sites
of low pO2.
 Cooperativity
Cooperativity refers to the allosteric effect of the action of one subunit on
other’s biological activity. If the action promotes the activity of other
subunits, it is said to be positive; if otherwise inhibits other subunit’s
activity, then negative co-operativity.
Hemoglobinopathies: refers to the pathological changes due to the
abnormal structure of Hb molecules. This disease is also called
“molecular disease”.

e.g. sickle-cell anemia is a disease resulted from the substitution of a

polar residue of the b-chain, Glu, by a hydrophobic one, Val. This
change causes aggregation of deoxy-HbS and hemolytic anemia.
 Sickle cell anemia (hemoglobin S disease)

 A genetic disorder of the blood caused by a single nucleotide

alteration (a point mutation) in the gene for -globin.
 The most common inherited blood disorder in the United States,
affecting 80,000 Americans.
 Recessive disorder, occuring in individuals who have inherited
two mutant genes (one from each parent) that code for
synthesis of the β chains of the globin molecules.
 characterized by lifelong episodes of pain (“crises”), chronic
hemolytic anemia with associated hyperbilirubinemia, and
increased susceptibility to infections.
 1. Amino acid substitution in Hb S β chains

The glutamate at position six has been replaced with

valine .
 2. Sickling and tissue anoxia:

The replacement of the charged glutamate with the nonpolar valine forms a protrusion on
the β-globin that fits into a complementary site on the β chain of another hemoglobin
molecule in the cell.
At low oxygen tension, deoxyhemoglobin S polymerizes inside the RBC, forming a
network of fibrous polymers that stiffen and distort the cell, producing rigid, misshapen
erythrocytes.
In lungs In peripheral tissues
OXY-STATE DEOXY-STATE
Aggregation of HbS

 

  
  
  
  
  


This interruption in the supply of oxygen leads to localized
anoxia (oxygen deprivation) in the tissue, causing pain and
eventually death (infarction) of cells in the vicinity of the
blockage.

Such sickled cells frequently

block the flow of blood in
the narrow capillaries
Biochemical Nature of SCA
 Primary Secondary Quaternary Red Blood
Function
Structure and Tertiary Structure Cell Shape
Structures
Normal Molecules do not
hemoglobin associate with one
1

Normal hemoglobin
another; each carries
2 oxygen.
3
4
5 
 subunit  10 m
6
7


Exposed
hydrophobic Molecules crystallize
Sickle-cell hemoglobin

1 Sickle-cell into a fiber; capacity

region hemoglobin
2 to carry oxygen is
reduced.
3
4
5 
6  10 m
7  subunit


 Medical/Biological Nature of SCA
 SCA is present at birth but does not manifest until 4
months after birth.

 Early diagnosis at birth can lengthen lifespan with

blood test.
 Using amniotic fluid or placenta tissue can be
used to diagnose SCA before birth.

 Signs & Symptoms Vary – can range from mild to

severe
 Linked to Anemia and Pain

 Numerous Complications
 Examples: Increased Risk of Infections, Splenic
Crisis, Acute Chest Syndrome, Multiple Organ
Failure
 Prevalence for SCA
Most Common Genetic Disorder in US – Affecting 70,000 –
100,000 Individuals
Primarily African Americans = 1 in 500
 Common in People from Africa, South and Central America,
Caribbean Islands, Mediterranean Islands, India, and Saudi Arabia
 Prevalence for SCA
Distribution of HbS allele
correlated to the distribution of
malaria – parasites that thrive in
normal RBCs cannot survive in
sickled RBCs.

Possible selective advantage of the heterozygous state

Hb S migrates more slowly toward the anode (positive
electrode) than does Hb A during electrophoresis at
alkaline pH (Figure 3.19).
This altered mobility of Hb S is a result of the absence
of the negatively charged glutamate residues in the two β
chains, thus rendering Hb S less negative than Hb A
 Section V
Physicochemical properties of protein
Physical properties

 Size
 Colloidal solutions
 Charge
 UV absorption
 Solubility
 A. Protein molecular size

Molecular weight:

Vary from 6000 to million Daltons (Da)

Protomeric proteins: 50 000 to 100 000 Da

Oligomeric proteins: > 100 000 Da

 B. Amphoteric properties: Protein charge
Proteins contain ionizable groups such as amino and carboxyl groups.
When the net charge is zero the pH is referred to as isoelectric point (pI) of
the protein.

(Zwitterion)

The pH at which the protein has zero net-charge is referred to as isoelectric

point (pI).
 C. Colloidal properties

 Solution（< 1 nm）
 Colloid（1 – 100 nm）
 Suspension(> 100 nm)
 Protein
 Particle size of 2~20 nm
 Protein solution has colloidal properties (high
viscosity, high absorption capacity, light distraction,
do not pass through a semipermeable membrane …
 D. UV absorption

 Ultraviolet light absorption of proteins results

from the residues that have peak absorbance at
280nm such as Trp, Tyr.

 Trp, Tyr have aromatic groups of resonance

double bonds.

 Proteins have a strong absorption at 280nm.

 This property is commonly used for

quantitative determination of proteins
 E. Solubility

Affected by the balance of hydrophobic and

hydrophilic amino acids on its surface

Charged amino acids play the most

important role in keeping the protein
soluble

Solubility determined by repulsion forces

among protein molecules and a hydration
water layer
 Influence of pH on protein solubility
 The proteins are least soluble at their isoelectric point (no net charge)
 The protein become increasingly soluble as pH is increased or
decreased away from the pI
 Influence of salt on protein solubility
 Positively and negatively charged small ions in solution can cluster around charged
side groups
 These ions can “screen” interacting side groups from each other
 Charged side groups of proteins can collect a cloud of ions called a counterion
atmosphere
 Extent of the counterion atmosphere depends on the ionic strength of the charged ions
in solution
 Influence of salt concentration on protein solubility

At low salt concentrations protein solubility

increases (salting-in): interactions between side
groups at the pI are screened which prevent
aggregation.

At high salt concentrations protein solubility

decreases (salting-out): salt ions bound to water
molecules and disrupt hydrolytic layer of
proteins.

157
 F. Denaturation of proteins
Protein denaturation ：unfolding and disorganization of the protein’s
secondary and tertiary structures, which are not accompanied by
hydrolysis of peptide bonds.
DENATURED STATE

• Loss of native conformation

Active protein • Altered secondary, tertiary or

quaternary structure

Protein loses structure • Disruption of disulfide bonds

and function Ovalbumin (covalent) and non-covalent bonds
(H-bond, ionic bond, hydrophobic
interaction
Denatured protein
• Peptide bonds are not affected
Denaturing agents include :
 heat,
 organic solvents,
 mechanical mixing,
 strong acids or bases,
 detergents,
 UV-light,
 High pressure,
 ions of heavy metals such as lead
and mercury.
Denaturation may, under ideal conditions, be
reversible, the protein refolds into its original native
structure when the denaturing agent is removed.

tu

Normal protein Denatured protein

 G. Color reactions:

Color reaction such as ninhydrin and biuret reactions.

Color reactions are also used for analysis of proteins.
 Questions

1. How dose primary structure of protein affect the function of protein?

2. What is the difference of oxygen binding between myoglobin and
hemoglobin? Explain the mechanism.
3. How dose protein denature affect protein function?