CHAPTER 5

PROTEIN FUNCTION
 Protein Function
• Fibrous proteins are structural elements of cells and
tissues depends on stable, long-term quaternary
interactions between identical polypeptide chains.

• The functions of many other proteins involve

interactions with a variety of different molecules.
Most of these interactions are transient, though they
may be the basis of complex physiological processes
such as oxygen transport and immune function.
• MYOglobin: MUSCLE, HEMOglobin: BLOOD
 Protein Function
• Ligand: A molecule bound reversibly by a protein. (any
molecule including protein).

• Binding site: the site on the protein where ligand binds ,

which is complementary to the ligand in size, shape, charge,
and hydrophobic or hydrophilic character.
• the interaction is specific: the protein can discriminate
among the thousands of different molecules in its
environment and selectively bind only one or a few.

• A given protein may have separate binding sites for several

different ligands.
 Protein Function
• Proteins are flexible. Changes in conformation
UNDERSTOOD, reflecting molecular vibrations and
small movements of amino acid residues throughout
the protein.

• A protein flexing in this way is sometimes said to

“breathe.”
Protein flexibility = Protein breathing

Changes in conformation reflects small movements of

amino acid throughout the protein.
 Protein Function
• The binding of a protein and ligand is often coupled to a conformational
change in the protein that makes the binding site more complementary to
the ligand, permitting tighter binding. The structural adaptation that
occurs between protein and ligand is called induced fit. In a multisubunit
protein, a conformational change in one subunit often affects the
conformation of other subunits.

• Interactions between ligands and proteins may be regulated, usually

through specific interactions with one or more additional ligands. These
other ligands may cause conformational changes in the protein that affect
the binding of the first ligand.

• Enzymes represent a special case of protein function. Enzymes bind and

chemically transform other molecules— they catalyze reactions. The
molecules acted upon by enzymes are called reaction substrates rather
than ligands, and the ligand-binding site is called the catalytic site or
active site.
Enzymes ligands = substrates.

Binding site = active site

 1. Storage and transport of ions and molecules

Myoglobin and Hemoglobin most studied, best understood

protein. 1st three dimensional revealed.

- O2 poorly soluble in aqueous soln. Diffusion through tissues is

ineffective over distances greater than a few millimeters.

- A.A. side chain not suitable to reversible binding of O2. transition

Metals (Iron) suitable for this function.

- Iron bound + sequestered in molecule making it less active(no ROS)

incorporated in heme group (prosthetic group).

i.e. Iron is often incorporated into a protein-bound prosthetic group

called heme.
Heme:
- A complex organic ring structure porphyrin bound to ferrous Fe 2+

- porphyrin consist of 4 pyrrole rings

- Coordinated nitrogen atoms ((which have an electron-donating

character) prevent
Fe2+ ( bind oxygen) ferric Fe3+ ( can not)
In free heme molecule irreversible conversion to Fe3+
In heme containing proteins, this reaction is prevented by sequestering of
the heme deep within the protein structure where access to the two open
coordination bonds is restricted

NO and CO carbon monoxide bind with greater affinity

toxic
Heme: iron has 6 coordination bonds

Organic ring structure

Porphyrin ring
Heme group viewed from the side:
2 coordination bonds to Fe2+ flat
porphoryin ring system.
One bound to O2 the other to
His (proximal His).

1. One of these two coordination bonds

is occupied by a side-chain nitrogen of
a His residue.

1. The other is the binding site for

molecular oxygen (O2)
Two histidine in Mb. One anchor the heme
group called proximal. The other help bind
oxygen called distal that reduce binding the
CO.
The Structure of Myoglobin
 Myoglobin: Simple oxygen-binding protein in all mammals primarily
Structure of myoglobin: in muscle tissue.

- Single polypeptide (153a.a)

- 8 cylinders A-H

-Nonhelical residues AB,CD …

segments that interconnect

-The heme pocket made up

largely from E and F helices.

- has histidine. His93 (the 93rd a.a residue ( the N: of the side chain of His attached to

Fe, from the N-terminal end of the myoglobin polypeptide sequence), is also called

His F8 (the 8th residue in alpha helix F)

• A reversible protein-ligand interaction
Myoglobin’s function depends on protein’s ability not only to bind
oxygen but also to release it.

Reversible binding of a protein P to a ligand L :

P + L  PL

Ka = [PL]
--------
[P] [L]
Ka= affinity of L to its P .
Ka= association constant = M-1
Kd = dissociation constant= 1/Ka = M
Kd is equivalent to the molar concentration of ligand at which half of the
available ligand-binding sites are occupied

Binding equilibrium. ( increase in L but ligand binding sites limited)

 (Theta) = binding sites occupied/ total binding sites
 = [PL]
---------
[PL]+[P]
The more tightly a protein binds a ligand  the lower [ligand] required
for the binding sites to be occupied lower Kd.
 Graphical representations of ligand binding

The fraction of ligand binding sites

occupied, θ plotted against [free ligand]

Kd = 1/Ka
The [L] at which half of the available ligand-
binding sites are occupied (at θ = 0.5)
corresponds to 1/Ka.
 Binding of Oxygen to Myoglobin

P50 is the oxygen concentration that equals Kd

O2 is a gas, conc. described by partial pressure of O2 expressed in kilopascals (Kpa).

O2 binds tightly to myoglobin with P50 of only 0.26KPa.

 Protein Structure Affects How Ligands Bind

• The interaction is greatly affected by protein structure and is

often accompanied by conformational changes. For example,
the specificity with which heme binds its various ligands is
altered when the heme is a component of myoglobin.

• Carbon monoxide binds to free heme molecules more than

20,000 times better than does O2, but it binds only about 200
times better when the heme is bound in myoglobin.

• The difference may be partly explained by steric hindrance.

When O2 binds to free heme, the axis of the oxygen molecule
is positioned at an angle to the Fe-O bond. In contrast, when
CO binds to free heme, the Fe, C, and O atoms lie in a straight
line
In arterial blood passing from the lungs through the heart to the
peripheral tissues, hemoglobin is about 96% saturated with oxygen.
In the venous blood returning to the heart, hemoglobin is only about
64% saturated.

Thus; each 100 mL of blood passing through a tissue releases about

one-third of the oxygen it carries
Myoglobin, with its hyperbolic binding curve for oxygen is relatively
insensitive to small changes in the concentration of dissolved oxygen
and so functions well as an oxygen-storage protein.

Hemoglobin, with its multiple subunits and O2-binding sites, is better

suited to oxygen transport.

Interactions between the subunits of a multimeric protein can permit a

highly sensitive response to small changes in ligand concentration.

Interactions among the subunits in hemoglobin cause conformational

changes that alter the affinity of the protein for oxygen. The
modulation of oxygen binding allows the O2-transport protein to
respond to changes in oxygen demand by tissues.
 Protein Structure Affects How Ligands Bind
• The binding of O2 to the heme in myoglobin also depends on
molecular motions, or “breathing,” in the protein structure.

• The heme molecule is deeply buried in the folded

polypeptide, with no direct path for oxygen to move from the
surrounding solute
• on to the ligand binding site.

• If the protein were rigid, O2 could not enter or leave the

heme pocket at a measurable rate. However, rapid molecular
flexing of the amino acid side chains produces transient
cavities in the protein structure, and O2 evidently makes its
way in and out by moving through these cavities.
 Structures of Myoglobin and the ß Subunit of Hemoglobin
structure of myoglobin and the β subunits of hemoglobin:
27a.a. identical
their structures are very similar to that of myoglobin, even though the amino acid
sequences of the three polypeptides are identical at only 27 positions

Most of the amino acids in hemoglobin form alpha helices, connected by short
non-helical segments. (Hemoglobin has no beta strands and no disulfide bonds).
Quaternary structure of hemoglobin show interaction with unlike
subunits. Hemoglobin consists of 2 alpha subunits and 2 beta subunits
to give a four chain structure.

The quaternary structure of hemoglobin features strong interactions

between unlike subunits. The α1β1 interface (and its α2β2 counterpart)
involves more than 30 residues, and its interaction is sufficiently strong

-Hemoglobin exists in 2 interchangeable structural states:

T (tense): stable when O2 not bound (-O2), Deoxy
R (relaxed): stable when O2 bound (+O2), Oxy
Dominant interactions between hemoglobin subunits

In this representation,α-
ubunits are light and β
subunits are dark.
The strongest subunit
interactions (highlighted)
occur between unlike
subunits.

When oxygen binds, the α1β1

contact changes little, but
there is a large change at the
α1β2 contact, with several ion
pairs broken
 The T R transition
The rotation of His HC3 residues

The transition from the T state (“tense=ion pairs”) to the R state (“relaxed” oxygenated) shifts the subunit
pairs substantially, affecting certain ion pairs. Most noticeably, the His HC3 residues at the carboxyl
termini of the subunits, which are involved in ion pairs in the T state, rotate in the R state toward the
center of the molecule, where they are no longer in ion pairs. Another dramatic result of the T to
R transition is a narrowing of the pocket between the beta subunits.
The T R transition:
The transition from the T state (“tense=ion pairs”) to the R state
(“relaxed” oxygenated) shifts the subunit pairs substantially,
affecting certain ion pairs
Positively charged side chains = blue.
Negatively charged side chains/ partners = red.
Binding to O2 conformational change to R
Shift in ion pairs mainly HC3 residue rotate toward molecule center.
Narrowing the pocket between β subunits.
Changes in conformation near heme on O2 binding to deoxyhemoglobin:
Shift in the position of F helix when heme binds O2 Heme gets a more planer
conformation.
Iron protrude toward the F helix more planner heme

1. The shift in the position of the F helix when heme binds O2 is thought to be one of
the adjustments that triggers the T to R transition.
2. Oxygen bind to HB in both states, but higher affinity in R state.
3. Heme assume Planar conformation: shifting position of His and attached F helix.
• His E7 (H bond) – coordinates O2
• His F8 coordinates Fe2+
• Two hydrophobic side chains on the O2
binding side of the heme help hole the heme
in place.Phe CD1, val E11 hydrophobic
bonding to porphyrin ring
• These side chains, Allow O2 to enter and exit.

http://www.youtube.com/watch?v=6AfRX6oh9-E
 Hb in RBC and oxygen binding
In arterial blood passing
from the lungs through TWO STATES: OXY HB and DEOXY HB
the heart to the peripheral
tissues, hemoglobin is
about 96% saturated with
oxygen. In the venous
blood returning to the
heart, hemoglobin is only
about 64% saturated.

Thus; each 100 mL of

blood passing through a
tissue releases about one-
third of the oxygen it
carries
 A sigmoid (cooperative) binding curve
1. A sigmoid binding curve
can be viewed as a Release O2 Retain O2
hybrid curve reflecting pO2=13.3kP
4kPa Case 1
a transition from a low-
affinity to a high-affinity
state. Hb case

2. Cooperative binding, as
manifested by a sigmoid
binding curve, renders
hemoglobin more
sensitive to the small
differences in O2 Case 2
concentration between the
tissues and the lungs,
allowing hemoglobin to
bind oxygen in the lungs
(where pO2 is high) and
release it in the tissues
(where pO2 is low).

EFFICIENT IN BINDING, AND EFFICIENT

IN UNLOADING OXYGEN
Hemoglobin must bind oxygen efficiently in the lungs, where the pO2 is
about 13.3 kPa, and release oxygen in the tissues, where the pO2 is
about 4 kPa.

Myoglobin, or any protein that binds oxygen with a hyperbolic

binding curve, would be ill-suited to this function.

A protein that binds O2 with high affinity would bind it efficiently in

the lungs but would not release much of it in the tissues. If the
protein bound oxygen with a sufficiently low affinity to release it in
the tissues, it would not pick up much oxygen in the lungs.
 O2 dissociation curve of hemoglobin  sigmoidal
O2 dissociation curve of myoglobin  hyperbolic

- Hemoglobin binds O2 efficiently in lungs (13.3 kPa) release it in

tissues (4 kPa).
If a Protein binds O2 with high affinity in lungs would not release it in
tissues.
If it binds O2 with low affinity to release it in tissues, it would not pick
up much O2 in lungs.
- O2 binding is both allosteric and cooperative.
As O2 binds to one site hemoglobin undergoes conformational change
affecting other binding sites.
- Conformational changes bw the T and R states mediated by subunit-
subunit interaction results in cooperative binding described by sigmoid
binding curve.
Allosteric protein: binding of a ligand to one site affects the binding
properties of another site on the same protein.
allos greek = other.
stereos = solid / shape.
Allosteric protein = having “other shapes” / conformation
induced by binding of ligand= modulator (inhibitor or activator).
Conformational changes more/less active.
Homotropic= normal ligand and modulator are identical.
Heterotropic= modulator other molecule than normal ligand.
One protein have several modulators homotropic/heterotropic.
O2 binding to hemoglobin : O2 ligand + homotropic modulator.
Two models for the inter conversions of inactive and active forms of
cooperative ligand binding proteins to multisubunit proteins.
a) Concerted model “all or none”: all subunits in the same
conformation. T state…R state (see equilibrium)

b) Sequential model: each individual subunit can be either ○(inactive) or

□ (active) large # of conformations is possible.
The concerted model assumes that the subunits of a
cooperatively binding protein are functionally identical, that
each subunit can exist in (at least) two conformations, and
that all subunits undergo the transition from one
conformation to the other simultaneously.

In this model, no protein has individual subunits in different

conformations. The two conformations are in equilibrium.
The ligand can bind to either conformation, but binds each
with different affinity. Successive binding of ligand
molecules to the low-affinity conformation (which is more
stable in the absence of ligand) makes a transition to the
high-affinity conformation more likely.
In the second model, the sequential model, ligand
binding can induce a change of conformation in an
individual subunit. A conformational change in one
subunit makes a similar change in an adjacent subunit,
as well as the binding of a second ligand molecule,
more likely.

There are more potential intermediate states in this

model than in the concerted model. The two models are
not mutually exclusive; the concerted model may be
viewed as the "all-or-none" limiting case of the
sequential model.
No explain for cooperate action

R state T state
When no O2 the
strucrure prefer the
T state state
T state

Limitation (this state

predominate when O2 BIND)

equlibrium

4 subunits= hemoglobin

IT REACH THE R STATE WHEN 4 O2 BIND

 A sigmoid (cooperative) binding curve
Release O2 Retain O2
pO2=13.3kP
4kPa Case 1

Hb case
R state

T state Case 2
Hemoglobin carries end products of respiration H+ and CO2 that stabilize T state.
CO2 + H2O ↔ H+ + HCO3-
carbonic anhydrase ( abundant in RBC)

CO2 insoluble in blood  formation of bubbles in blood and tissues if not converted to bicarbonate.
CO2 Hydration to bicarbonate increase [H+ ]  decrease in pH .
Affinity of hemoglobin to O2 ↓ as pH ↓ in peripheral tissue.
Hemoglobin transports about 40% of the total H+ and 15% to 20% of the CO2 formed in the tissues to the lung
and the kidneys. (The remainder of the H+ is absorbed by the plasma's bicarbonate buffer; the remainder of the
CO2 is transported as dissolved HCO3- and CO2).
In lungs capillaries CO2 is excreted.
in the capillaries of the lung, as CO2 is excreted and the blood pH consequently rises, the affinity of
hemoglobin for oxygen increases and the protein binds more O2
decrease PH…..release O2 while increase ….bound O2
Bohr effect = effect of pH and [CO2 ] on O2 binding and release.
Hb + O2 ↔ HbO2 (oxygen binds to iron in Heme).
HHb + O2 ↔ HbO2 + H+ (proton binds to any of the a.a).
His149 (His HC3) of B subunit when protonated ion pair- Asp94  stabilize T state in deoxyhemoglobin
Effect of pH on O2
binding to hemoglobin.
pH of blood in lungs =
Higher [H+]
7.6 Lower
affinity to
In tissues 7.2 O2

Lung/tissue
O/H+

O2-saturation curve of hemoglobin is influenced by the H+ concentration.

Both O2 and H+ are bound by hemoglobin, but with inverse affinity. When the oxygen
concentration is high, as in the lungs, hemoglobin binds O2 and releases protons. When
the oxygen concentration is low, as in the peripheral tissues, H+ is bound and O2 is
released
CO2 binds hemoglobin inversely to O2
Binds as a carbamate group to the N-terminal end of
each globin chain  carbaminohemoglobin.

H+ produced contribute to Bohr effect.

Oxygen binding to hemoglobin is modulated by 2,3-bisphosphoglycerate which binds to and
stabilize T state.
Unlike O2, only one molecule of BPG is bound to each hemoglobin tetramer. BPG binds at a site
distant from the oxygen-binding site

Heterotropic allosteric modulation (effect):

Reduce affinity of Hb to O2.

- Physiological adaptation to
the lower pO2 in high altitudes.
[BPG ] ↑ , affinity to O2 ↓ .

- O2 delivered to tissues >> 40%.

Effect of BPG on hemoglobin
binding to O2 :
BPG conc in normal human
blood= 5mM at sea level,
8nm at high altitudes.
Hemoglobin binds O2 tightly
when BPG is entirely absent,
At sea level hemoglobin is
nearly saturated with O2 in lungs.
But only 60% in other tissues.
At high altitudes O2 delivery
declines by one fourth.
Regulation of O2 binding to hemoglobin by BPG
important in fetal development:
Fetus has α2γ2 hemoglobin.
This tetramer has ↓ affinity to BPG than adult
hemoglobin  ↑ affinity to O2.
Fetus can extract O2 from maternal blood.
Normal uniform, cup shaped erythrocytes:
Sickle cell anemia:
- a genetic disease caused by a single a.a substitution ( Glu6 –ve
charge by  Val6 no charge) in each ß chain of hemoglobin.
- The change produces a hydrophobic patch on the surface of
hemoglobin that causes the molecules to aggregate (sticky) into
fibers bundles when hemoglobin S is deoxygenated.

This homozygous condition results in serious medical

complications.
Erythrocytes in sickle cell anemia:
Fewer + abnormal + immature + some blade shaped.
Normal and sickle cell hemoglobin:
A single a.a change ( Glu 6Val 6 ) in β chains
Deoxyhemoglobin S has a
hydrophobic patch on its surface

Causing the molecules to be sticky

and aggregate into strands that align
into insoluble fibers.
Insoluble fibers responsible for
deformed shape.

Normal hemoglobin soluble

Hemoglobin S insoluble when
deoxygenated.
 Effect of Temperature
• hyperthermia causes a rightward shift, while
hypothermia causes a leftward shift
• Increasing temperature will weaken and
denature the bond between an oxygen and a
hemoglobin which in turn decreases the
concentration of the oxyhemoglobin
 Right/Left shift
• Right shift • Left shift
1. Decrease pH(increase • Higher affinity to
H+) oxygen
2. Increase CO2 • Increase pH
3. Increase Temp • Decrease CO2
4. Increase BPG • Decrease Temp
5. Hypoxia • Decrease BPG
• Fetal Hb.