BIOCHEMISTRY (I)

LIFS2210

Protein Function and Evolution

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 The Four Levels of Protein Structure

amino acid linear sequence

regions of regularly repeating conformations of the

peptide chain, such as -helices and -sheets

describes the shape of the fully

folded polypeptide chain arrangement of two or more polypeptide
chains into multisubunit molecule
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Role of the globins in oxygen transport
and storage

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 1. Structures of Myoglobin and Hemoglobin

• Myoglobin (Mb) - monomeric protein that

facilitates the diffusion of oxygen in vertebrates
Myoglobin is composed of 8  helices

• Hemoglobin (Hb) - tetrameric protein that carries

oxygen in the blood
Hb is an 22 tetramer (2  globin subunits, 2  globin subunits)
Each globin subunit is similar in structure to myoglobin
Each subunit has a heme group
The  chain has 7  helices,  chain has 8  helices
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Myoglobin and Hemoglobin

myoglobin hemoglobin
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 2. Oxygen Binding to Mb and Hb

Oxygen Binds Reversibly to Heme

• Oxymyoglobin - oxygen bearing myoglobin

• Deoxymyoglobin - oxygen-free myoglobin

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 Heme Fe(II)-protoporphyrin IX

• Heme of Mb and Hb binds oxygen for transport

• Heme consists of a tetrapyrrole ring system called
protoporphyrin IX complexed with iron

Protoporphyrin IX Ferroprotoporphyrin IX

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The geometry of iron coordination in
oxymyoglobin

• Oxygen
• His-93 and
His-64

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 Oxygen-binding site of
oxymyoglobin

• Octahedral geometry of
coordination complex (six
ligands around iron)
• His-93 (proximal histidine)
liganded to Fe
• His-64 (distal histidine)

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 Oxygen Binding to Mb

• Heme occupies a hydrophobic cleft formed by

three a helices and two loops
• Heme prosthetic group binds oxygen
• His-93 is complexed to the iron atom, and His-64 forms a
hydrogen bond with oxygen
• Oxygen is coordinated between the iron and the imidazole
sidechain of His-64
• Interior of Mb almost all hydrophobic amino acids

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 3. Oxygen-Binding Curves of
Myoglobin and Hemoglobin

Comparison of O2-
binding to Mb and Hb
θ

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 Oxygen-binding curves
• Curves show reversible binding of O2 to Mb and Hb
• Fractional saturation (θ, % saturation) is plotted versus the
partial pressure of oxygen, pO2 (oxygen concentration)
• The shape of the Hb curve shows a positive cooperativity in
the binding of 4 O2 molecules
(i.e. the O2 affinity of Hb increases as each O2 molecule is
bound)
• Mb-O2 binding curve is hyperbolic, indicating a single equilibrium
constant for binding O2
• Hb-O2 binding curve is sigmoidal, and reflects the binding of 4
molecules of O2, one per each heme group
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 4. Hemoglobin is an Allosteric Protein

• Oxygen binding and release from Hb are

regulated by allosteric interactions
• Allosteric effectors (modulators) bind to a
protein at a site separate from the functional
binding site (may be activators or inhibitors)
• The activity of an allosteric protein is
regulated by allosteric effectors

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Two conformations of hemoglobin: T and R

• Active (R state, relaxed state) and inactive

(T state, tense state) forms are in rapid
equilibrium in allosteric proteins
• Binding of substrates and allosteric activators
stabilize the R state and shift the equilibrium
in the R direction
• Allosteric inhibitors stabilize the T state and
shift the equilibrium in the T direction

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 Oxygen-binding curves

Oxygen binding of
the R (high-affinity)
and T (low affinity)
forms of Hb θ

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The change in hemoglobin quaternary
structure during oxygenation

Side view

Top view
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Mechanism of the T - R transition in
hemoglobin

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 2,3-bisphospho-D-glycerate (2,3-BPG)
• 2,3-BPG is an allosteric effector of Hb
• 2,3-BPG lowers the affinity of deoxyHb for oxygen
(raises the P50 of Hb from ~12 to ~26 torr)
• Negatively charged 2,3-BPG is bound to six
(+) charged groups of deoxyhemoglobin

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 Binding of 2,3-BPG to
deoxyhemoglobin
• (-) Charges on 2,3-BPG pair with (+) charges lining
the central cavity, stabilizing the deoxyHb form
• -Subunits (brown), -subunits (blue), heme groups
(red)

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 5. Bohr effect in hemoglobin

• A pH drop in the blood in the

capillaries lowers the oxygen
affinity of Hb, allowing even
more efficient release of the
last traces of oxygen.
• The response of hemoglobin
to changes in pH is called the
Bohr effect.
• Lowering the pH decreases
the affinity of Hb for oxygen
• Mb has little Bohr effect.

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 Carbon dioxide transport

• Carbon dioxide is transported

from the tissues to the lungs in
two ways:
(1) Dissolved bicarbonate ions
(2) Carbamate adducts of
hemoglobin (N-terminal globin
residues react with CO2 to
form carbamates)

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Combined
effects of CO2
and BPG on
oxygen binding
by hemoglobin

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 6. Immunoglobulins

• Variability in structure yields versatility

in binding

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 Antibodies (immunoglobulins)

• Vertebrate immune systems

synthesize protein antibodies
(immunoglobulins) to eliminate
bacteria, viruses, other foreign
substances
• Antibodies specifically
recognize and bind antigens
• Antibodies are synthesized by
lymphocytes (white blood
cells)

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 Antibodies Bind Specific Antigens

•Heavy chains (green)

•Light chains (blue)

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Clonal selection theory of the immune response

1. B stem cells in the bone marrow differentiate to become B lymphocytes, each

producing a slightly different type of immunoglobulin molecule, each type with
a binding site that will recognize specific molecular shapes. These
immunoglobulins, or antibodies, are attached to the cell membrane and
exposed on the outer surfaces of the B lymphocytes.
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Clonal selection theory of the immune response

2. Binding of an antigen to one of these antibodies stimulates the cell carrying it to

replicate, generating a clone (a collection of cells with identical genetic
information). This primary response is aided by a special class of T cells called
helper T cells. If a helper T cell recognizes a bound antigen, it binds to the
appropriate B lymphocyte and stimulates B-cell reproduction.
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Clonal selection theory of the immune response

3. Plasma B cells produce soluble antibodies, which are excreted into the circulatory
system. These antibodies have the same antigen binding sites as the surface
antibodies of the B lymphocyte from which the plasma cells arose, but they lack
the hydrophobic tail that bound the surface antibodies to the lymphocyte
membrane.
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 7. Coding and noncoding regions of
the  hemoglobin gene
Flow of information: from nucleic acids to proteins
DNA RNA Protein

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 From Gene to Function

x 5 to 50
~25,000 Genes > 1,000,000 Proteins functional
links per protein
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Conclusions of the genome projects

We have about the same number of genes as

fish and plants, and not that many more genes
than worms and flies.

Fugu rubripes (pufferfish): 31,000 to 38,000

Arabidopsis thaliana (thale cress): 26,000
Caenorhabditis elegans (worm): 19,000
Drosophila melanogaster (fly): 13,000

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 What is proteomics?

• Proteome
• "The entire PROTEin complement expressed by a genOME,
or by a cell or tissue type."
Marc Wilkins & Keith Williams. BioTechnology 14 (1996) 61-65

• Proteomics - study of large sets of proteins, such as the entire

complement of proteins produced by a cell under defined
conditions.

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Why Proteomics?

• Genomics can tell us

events that may happen in
cells.

• Proteomics tells us when,

where and how these
predicted events happen in
the cells.

• Proteomics bridges the

knowledge gap between
genes and what actually
happens in the cells.

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