David L. Nelson and Michael M.

Cox

LEHNINGER
PRINCIPLES OF BIOCHEMISTRY
Sixth Edition

CHAPTER 5
Protein Function

© 2013 W. H. Freeman and Company

 Internal III on
01-11-2018 Thursday
3.00 pm
Reversible Binding of a Protein to a Ligand: Oxygen-Binding Proteins
Oxygen Can Bind to a Heme Prosthetic Group

Heme
The heme group
viewed from the
side
Globins Are a Family of Oxygen-Binding Proteins

Myoglobin Has a Single Binding Site for Oxygen

Myoglobin
M 16,700
r

153 AAs
Protein-Ligand Interactions Can Be Described Quantitatively
Binding of oxygen to myoglobin
Which protein (X or Y) has a greater affinity for ligand A?

What is the dissociation constant, Kd, for each protein?

 Protein Structure Affects
How Ligands Bind

Ligand binding to His64

the heme of
myoglobin

Kd for CO binding to free

heme is >20,000 times lower
than that for O , but it binds
2

only about 200 times better

than O when the heme is
2

bound in myoglobin His93

Hemoglobin Transports Oxygen in Blood
Hemoglobin Subunits Are Structurally Similar to Myoglobin
Comparison of the structure of myoglobin and the
beta subunit of hemoglobin

M 64,500
r

 chain: 141 AAs

β chain: 146 Aas
The amino acid sequences of
whale myoglobin and the
and  and β chains of human
hemoglobin
 >30 residue interaction
betn 1 β1 or 2 β2.

19 residue interaction
betn 1 β1 and 2 β2.

Dominant interactions between hemoglobin subunits. (PDB ID 1HGA) In this representation,  subunits
are light and β subunits are dark. The strongest subunit interactions (highlighted) occur between unlike
subunits. When oxygen binds, the 1β1 contact changes little, but there is a large change at the 1β2
contact, with several ion pairs broken.
Some ion pairs that stabilize the T
state of deoxyhemoglobin.
(a) Interactions between the ion
pairs His HC3 and Asp FG1 of the
β subunit (blue) and between Lys
C5 of the  subunit (gray) and His
HC3 (its -carboxyl group) of the β
subunit are shown with dashed
lines. (Recall that HC3 is the
carboxyl-terminal residue
of the β subunit.)

(b) Interactions between

these ion pairs, and
between others not shown
in (a), are schematized in
this representation
of the extended polypeptide
chains of hemoglobin.
Hemoglobin is an α2β2 tetramer that changes
conformation from a T state to R state upon
binding oxygen

Narrowing of pockets in β-subunits, Rotation of His HC3 toward center

 Changes in conformation near heme on O2 binding to
deoxyhemoglobin.

Iron is 0.4 to 0.6 A away from plane in deoxy-Hb,

HbO2: 0.09 A (α) and 0.06 A (β)
Oxygen binding by hemoglobin exhibits a sigmoidal shape, indicating
cooperative binding of oxygen and a shift from the R to the T state.
A Hill plot indicates the degree of cooperativity for
oxygen binding to hemoglobin
 Models for cooperative binding of oxygen to hemoglobin
The concerted model versus the sequential model

Jacques Monod, Jeffries Wyman,

and Jean-Pierre Changeux
Sequential model: Daniel Koshland
 Effect of pH on oxygen binding to hemoglobin

Bohr effect

His146 (His HC3) of the

β subunits.
Forms ion pairs—to
Asp94 (Asp FG1)

Protonation of the amino-terminal

residues of the  subunits, certain
other His residues, and perhaps
other groups has a similar effect.
Carbon dioxide binds to the amino terminus of
each globin chain of hemoglobin
Contributes to the Bohr effect

Carbaminohemoglobin

additional salt bridges help to stabilize the T state

and promote the release of oxygen.
 Oxygen binding to hemoglobin is regulated by
2,3-bisphosphoglycerate
Heterotropic allosteric
modulation:

HbBPG + O2 HbO2 + BPG

The BPG concentration
in erythrocytes also
increases in people
suffering from hypoxia,
lowered oxygenation of
peripheral tissues
due to inadequate
functioning of the lungs or
circulatory system.
 One BPG molecule binds to the hemoglobin tetramer
T state R state

Narrowing of pockets in β-subunits

Precludes BPG binding
Cavity between the  subunits
Ionic interaction, T state stabilized The fetus synthesizes  subunits
rather than  subunits, forms α22 Hb.
 Sickle-Cell Anemia Is a Molecular Disease of Hemoglobin

~500 genetic
variants of Hb

the allele for sickle-

cell hemoglobin from
both parents

Hb S deoxygenated
is insoluble, forms
polymer

uniform, cup-shaped spiny or sickle-shaped

Sickle cell anemia: long, thin, sickle-shaped erythrocytes

 Glu6 to Val in the two 
subunits.

Val creates a “sticky”

hydrophobic contact points on
the outer surface of Hb.

Sticky spots of deoxyHb S

associate abnormally with each
other, form the long, fibrous
aggregates characteristic of this
disorder.
 The Immune Response Features a Specialized Array of Cells
and Proteins

Leukocytes (white blood cells), macrophages and lymphocytes

Two complementary systems, the humoral and cellular immune systems

Humoral (Latin humor, “fluid”) immune system directed at bacterial

infections and extracellular viruses (those found in the body fluids),
but can also respond to individual foreign proteins.

Immune response by soluble proteins called antibodies or

immunoglobulins, Ig. 20% of blood protein.

Produced by B lymphocytes, or B cells (development in the bone marrow)

Cellular immune system destroys host cells infected by viruses

and also destroys some parasites and foreign tissues
T lymphocytes, or T cells (development occur in the thymus))

known as cytotoxic T cells (TC cells, also called killer T cells)

Infected cells or parasites recognition: proteins called T-cell

receptors on the surface of Tc cells.

Helper T cells (TH cells): produce soluble signaling proteins called

cytokines, which include the interleukins.

TH cells interact with macrophages.

Indirectly participate in the destruction of infected cells and

pathogens, stimulating the selective proliferation of those TC and B
cells that can bind to a particular antigen.

This process, called clonal selection, increases the number of

immune system cells that can respond to a particular pathogen.
Humans are capable of producing >108 different antibodies
with distinct binding specificities.

Antigen: Any molecule or pathogen capable of eliciting an

immune response. ….. be a virus, a bacterial cell wall,
or an individual protein or other macromolecule.

An individual antibody or T-cell receptor binds only a particular

molecular structure within the antigen, called its antigenic
determinant or epitope.

Molecules of Mr < 5000 are generally not antigenic.

Haptens, small molecules are covalently attached to large proteins

in the laboratory, they can be used to elicit an immune response.
Antibodies Have Two Identical
Antigen-Binding Sites
 Heavy chains: α, , , , and  for
IgA, IgD, IgE, IgG, and IgM,
respectively.
 and  light chains.

IgD and IgE are

similar to that of IgG

IgA, found principally

in secretions such as saliva,
tears, and milk, can be
a monomer, dimer, or trimer.
IgE plays an important role in the allergic response, interacting
with basophils (phagocytic leukocytes) in the blood
Antibodies Bind Tightly and Specifically to Antigen

antibody-antigen interaction is quite strong,

characterized by Kd values as low as 10-10 M
The Antibody-Antigen Interaction Is the Basis for a Variety of Important
Analytical Procedures
Polyclonal antibodies:
produced by many
different B lymphocytes
responding to one antigen

Monoclonal antibodies:
synthesized by a
population of identical B
cells (a clone) grown in
cell culture

ELISA (enzyme-linked
immunosorbent assay)